Morphic forms of hexadecyloxypropyl-phosphonate esters and methods of synthesis thereof

ABSTRACT

The disclosure describes methods of synthesis of phosphonate ester compounds. The methods according to the disclosure allow for large-scale preparation of phosphonate ester compounds having high purity and stability. Also disclosed are morphic forms of phosphonate ester compounds.

RELATED APPLICATIONS

This application claims priority to, and the benefit of, U.S. patentapplication Ser. No. 14/512,335, filed Oct. 10, 2014, which claimspriority to U.S. Provisional Application No. 61/904,857, filed Nov. 15,2013, the entire content of which is incorporated herein by reference inits entirety.

TECHNICAL FIELD

This disclosure relates to solid crystalline forms of Phosphonic acid,[[(S)-2-(4-amino-2-oxo-1(2H)-pyrimidinyl)-1-(hydroxymethyl)ethoxy]methyl]mono[3-(hexadecyloxy)propyl]ester, and relatedcompositions and methods. The methods provided in the present disclosureprovide synthesis of morphic forms of Compound 1 with high purity, highyield, and high stability, and crystals thereof.

BACKGROUND OF THE INVENTION

In the pharmaceutical drug development there is a need for reproduciblemanufacturing methods for obtaining pharmaceutically active ingredientsin chemically and morphologically pure form. Obtaining thepharmaceutically active ingredient in homogeneous solid state is aprecondition for complying with the requirements of the industrialmanufacture of finished dosage forms. Solid forms of the same activeingredient having different morphology may exhibit significantdifferences in the rate of dissolution, bioavailability and chemicalstability. From the viewpoint of industrial chemical and pharmaceuticaltechnology, it is important, therefore, that different solid forms of anactive ingredient can possess significantly different properties withregard to the operations of the technology, e.g. rate of filtration ordrying, solubility, behavior during tableting. The properties mentionedhere have a direct impact on the efficiency, economy, reproducibilityand complexity of the industrial manufacturing process and may result ina morphologically homogeneous product.

It is generally accepted that crystalline forms of pharmaceuticallyactive ingredients possess improved chemical stability as compared tothe amorphous form. Due to the different decomposition processes duringthe manufacture and shelf-life of the finished dosage form, thisdifference in stability is of general importance. Therefore,manufacturers of medicinal products prefer to use crystalline forms ofthe active ingredients during pharmaceutical development.

The polymorphism of a pharmaceutically active ingredient can beexploited in several ways. For example, using a crystalline form havingsuitable stability and impurity profile (purity) for the manufacture ofa finished dosage form is of paramount importance. It is alsosignificant that a crystalline active ingredient should have appropriateproperties for manipulations of large-scale manufacturing andpharmaceutical technology on an industrial scale. However, differentproperties of polymorphs, e.g. dissolution rate, particle size etc. canalso be exploited during the design of different finished dosage forms.A polymorph having lower dissolution rate may contribute to theproperties of a delayed release dosage form, while the skilled personmay appreciate a form having higher solubility or higher dissolutionrate during the formulation of an immediate release dosage form.Moreover, it is not routine practice in the art to make a specificpolymorph. The present invention is directed at providing uniquepolymorphs of Compound 1 with desirable features.

SUMMARY OF THE INVENTION

The present disclosure, in part, provides a method for manufacturingindustrial scale solid forms (e.g. crystalline forms) of phosphonicacid, [[(S)-2-(4-amino-2-oxo-1(2H)-pyrimidinyl)-1-(hydroxymethyl)ethoxy]methyl]mono[3-(hexadecyloxy)propyl]ester (Compound 1). In someembodiments, the present invention provides methods of synthesizingCompound to provide it in high purity and high yield. In someembodiments, the crystalline forms of Compound 1 synthesized by themethod of the present disclosure are stable as compared to the amorphousform.

It is an object of this disclosure to provide novel process for thepreparation of solid forms (e.g., crystalline forms) of phosphonic acid,[[(S)-2-(4-amino-2-oxo-1(2H)-pyrimidinyl)-1-(hydroxymethyl)ethoxy]methyl]mono[3-(hexadecyloxy)propyl]ester (“Compound 1”) in goodyield, in large amounts, and with desired purity. Provided herein arenovel solid forms (e.g., crystalline forms) of phosphonic acid,[[(S)-2-(4-amino-2-oxo-1(2H)-pyrimidinyl)-1-(hydroxymethyl)ethoxy]methyl]mono[3-(hexadecyloxy)propyl]ester (“Compound 1”).

One embodiment is directed to polymorph or morphic Form II (the terms“polymorph” or “morphic Form” are used interchangeably in thisdisclosure). In some embodiments, a composition comprising the morphicForm II of the present disclosure is substantially free of impurities,i.e., not a sufficient amount of impurities are present in the sample ofForm II. In some embodiments, a composition comprising the morphic FormII is substantially free of amorphous Compound 1. The embodiments of thepresent disclosure also provide that the Form II is substantially freeof hydrate form. In some embodiments, a composition comprising Form IIis anhydrous.

In some embodiments, a composition comprising the morphic Form II of thepresent disclosure is less than about 3 mg/mL soluble in 1:1methanol:water ratio at room temperature and less than about 14 mg/mLsoluble at about 63° C. A composition comprising morphic Form II of thepresent disclosure can have an X-ray diffraction pattern includingprominent peaks at about 2.81 and about 5.63 degrees 2θ.

In one embodiment, a composition comprising the morphic Form II of thecompound of the present embodiment is characterized by an X-raydiffraction pattern substantially similar to that set forth in FIG. 1,FIG. 7, FIG. 13, FIG. 14, FIG. 20, FIG. 21, or FIG. 25, indexingsubstantially similar to that set forth in FIG. 2, and/or have a DSCThermogram substantially similar to that set forth in FIG. 4, FIG. 15,FIG. 16, or FIGS. 22-24.

One embodiment of the present disclosure provides a morphic Form II of acompound having Formula II or III characterized by an X-ray diffractionpattern with two or more (e.g., three or more, four or more, five ormore, six or more, seven or more, eight or more, nine or more, or ten ormore) peaks expressed in degrees 2θ (±0.2) selected from 2.81, 5.63,11.30, 12.05, 13.22, 13.45, 13.81, 14.32, 14.92, 15.64, 16.25, 16.41,17.00, 17.67, 17.87, 18.15, 18.35, 18.50, 19.00, 19.57, 19.85, 20.22,20.96, 21.06, 21.89, 22.76, 23.70, 23.95, 24.32, 24.70, 25.54, 26.12,26.52, 26.81, 27.07, 27.48, 27.71, 29.11, 29.36, and 29.61, or having anX-ray diffraction pattern substantially similar to that set forth inFIG. 1, FIG. 7, FIG. 13, FIG. 14, FIG. 20, FIG. 21, or FIG. 25. In oneembodiment, a composition comprising the morphic Form II has indexingsubstantially similar to that set forth in FIG. 2 and/or a DSCThermogram substantially similar to that set forth in FIG. 4, FIG. 15,FIG. 16 or FIGS. 22-24. In some embodiments, a composition comprisingthe morphic Form II is characterized by ¹H NMR substantially similar tothat set forth in FIG. 6.

The present disclosure provides, morphic Form II of a compound ofFormula II or III (or a pharmaceutically acceptable salt thereof)characterized by an X-ray diffraction pattern with two or more (e.g.,three or more, four or more, five or more, six or more, seven or more,eight or more, nine or more, or ten or more) peaks expressed in degrees2θ (±0.2) selected from 2.81, 5.63, 11.30, 12.05, 13.22, 13.45, 13.81,14.32, 14.92, 15.64, 16.25, 16.41, 17.00, 17.67, 17.87, 18.15, 18.35,18.50, 19.00, 19.57, 19.85, 20.22, 20.96, 21.06, 21.89, 22.76, 23.70,23.95, 24.32, 24.70, 25.54, 26.12, 26.52, 26.81, 27.07, 27.48, 27.71,29.11, 29.36, and 29.61, or having an X-ray diffraction patternsubstantially similar to that set forth in FIG. 1, FIG. 7, FIG. 13, FIG.14, FIG. 20, FIG. 21, or FIG. 25, and/or characterized by ¹H NMRsubstantially similar to that set forth in FIG. 6, administered at adose of about 100 mg twice a week or at a dose of about 200 mg once aweek. In one embodiment, a composition comprising the morphic Form IIhaving indexing substantially similar to that set forth in FIG. 2 and/ora DSC Thermogram substantially similar to that set forth in FIG. 4, FIG.15, FIG. 16 or FIGS. 22-24 is administered at a dose of about 100 mgtwice a week or at a dose of about 200 mg once a week.

The present disclosure provides morphic Form II of a compound of FormulaII or III (or a pharmaceutically acceptable salt thereof) characterizedby an X-ray diffraction pattern with two or more (e.g., three or more,four or more, five or more, six or more, seven or more, eight or more,nine or more, or ten or more) peaks expressed in degrees 2θ (±0.2)selected from 2.81, 5.63, 11.30, 12.05, 13.22, 13.45, 13.81, 14.32,14.92, 15.64, 16.25, 16.41, 17.00, 17.67, 17.87, 18.15, 18.35, 18.50,19.00, 19.57, 19.85, 20.22, 20.96, 21.06, 21.89, 22.76, 23.70, 23.95,24.32, 24.70, 25.54, 26.12, 26.52, 26.81, 27.07, 27.48, 27.71, 29.11,29.36, and 29.61, or having an X-ray diffraction pattern substantiallysimilar to that set forth in FIG. 1, FIG. 7, FIG. 13, FIG. 14, FIG. 20,FIG. 21, or FIG. 25, and/or characterized by ¹H NMR substantiallysimilar to that set forth in FIG. 6, administered at a dose of about 1-4mg/kg (e.g., about 1.0-1.1 mg/kg, about 1.1-1.2 mg/kg, about 1.2-1.3mg/kg, about 1.3-1.4 mg/kg, about 1.4-1.5 mg/kg, about 1.5-1.6 mg/kg,about 1.6-1.7 mg/kg, about 1.7-1.8 mg/kg, about 1.8-1.9 mg/kg, about1.9-2.0 mg/kg, about 2.0-2.1 mg/kg, about 2.1-2.2 mg/kg, about 2.2-2.3mg/kg, about 2.3-2.4 mg/kg, about 2.4-2.5 mg/kg, about 2.5-2.6 mg/kg,about 2.6-2.7 mg/kg, about 2.7-2.8 mg/kg, about 2.8-2.9 mg/kg, about2.9-3.0 mg/kg, about 3.0-3.1 mg/kg, about 3.1-3.2 mg/kg, about 3.2-3.3mg/kg, about 3.3-3.4 mg/kg, about 3.4-3.5 mg/kg, about 3.5-3.6 mg/kg,about 3.6-3.7 mg/kg, about 3.7-3.8 mg/kg, about 3.8-3.9 mg/kg, or about3.9-4.0 mg/kg).

In some embodiments, a composition comprising the morphic Form II havingindexing substantially similar to that set forth in FIG. 2 and/or a DSCThermogram substantially similar to that set forth in FIG. 4, FIG. 15,FIG. 16 or FIGS. 22-24 is administered at a dose of about 1-4 mg/kg(e.g., about 1.0-1.1 mg/kg, about 1.1-1.2 mg/kg, about 1.2-1.3 mg/kg,about 1.3-1.4 mg/kg, about 1.4-1.5 mg/kg, about 1.5-1.6 mg/kg, about1.6-1.7 mg/kg, about 1.7-1.8 mg/kg, about 1.8-1.9 mg/kg, about 1.9-2.0mg/kg, about 2.0-2.1 mg/kg, about 2.1-2.2 mg/kg, about 2.2-2.3 mg/kg,about 2.3-2.4 mg/kg, about 2.4-2.5 mg/kg, about 2.5-2.6 mg/kg, about2.6-2.7 mg/kg, about 2.7-2.8 mg/kg, about 2.8-2.9 mg/kg, about 2.9-3.0mg/kg, about 3.0-3.1 mg/kg, about 3.1-3.2 mg/kg, about 3.2-3.3 mg/kg,about 3.3-3.4 mg/kg, about 3.4-3.5 mg/kg, about 3.5-3.6 mg/kg, about3.6-3.7 mg/kg, about 3.7-3.8 mg/kg, about 3.8-3.9 mg/kg, or about3.9-4.0 mg/kg).

The present disclosure provides, morphic Form H of a compound of FormulaII or III (or a pharmaceutically acceptable salt thereof) characterizedby an X-ray diffraction pattern substantially similar to that set forthin FIG. 18, administered at a dose of about 100 mg twice a week or at adose of about 200 mg once a week. In one embodiment, a compositioncomprising the morphic Form H having indexing substantially similar tothat set forth in FIG. 3 and/or a DSC Thermogram substantially similarto that set forth in FIG. 17 is administered at a dose of about 100 mgtwice a week or at a dose of about 200 mg once a week.

In some embodiments, a composition comprising the morphic Form H ischaracterized by an X-ray diffraction pattern substantially similar tothat set forth in FIG. 18, having indexing substantially similar to thatset forth in FIG. 3, and/or a DSC Thermogram substantially similar tothat set forth in FIG. 17 is administered at a dose of about 1-4 mg/kg(e.g., about 1.0-1.1 mg/kg, about 1.1-1.2 mg/kg, about 1.2-1.3 mg/kg,about 1.3-1.4 mg/kg, about 1.4-1.5 mg/kg, about 1.5-1.6 mg/kg, about1.6-1.7 mg/kg, about 1.7-1.8 mg/kg, about 1.8-1.9 mg/kg, about 1.9-2.0mg/kg, about 2.0-2.1 mg/kg, about 2.1-2.2 mg/kg, about 2.2-2.3 mg/kg,about 2.3-2.4 mg/kg, about 2.4-2.5 mg/kg, about 2.5-2.6 mg/kg, about2.6-2.7 mg/kg, about 2.7-2.8 mg/kg, about 2.8-2.9 mg/kg, about 2.9-3.0mg/kg, about 3.0-3.1 mg/kg, about 3.1-3.2 mg/kg, about 3.2-3.3 mg/kg,about 3.3-3.4 mg/kg, about 3.4-3.5 mg/kg, about 3.5-3.6 mg/kg, about3.6-3.7 mg/kg, about 3.7-3.8 mg/kg, about 3.8-3.9 mg/kg, or about3.9-4.0 mg/kg).

In some embodiments, Form II is distinguished from Form H. For instance,FIG. 25 shows a comparison of the X-ray powder diffraction spectra fortwo samples of Form II (top two spectra) and one sample of Form H(bottom sample). As seen from the comparison, Form II is different fromForm H in that it has significant peaks at about, for instance, 24.8° 2θand 22.9° 2θ. In contrast, in some embodiments Form II does not havesubstantial peaks that are seen in Form H, for instance at about 24.1°2θ, 21.5° 2θ, 20.9° 2θ, and 12.6 ° 2θ.

In some embodiments the present disclosure provides Compound 1 Form II(or a pharmaceutically acceptable salt thereof) formulated as apharmaceutical composition (see Tables 11 and 12). In one embodiment,Compound 1 Form II (or a pharmaceutically acceptable salt thereof) isformulated as a tablet of formulation 1 (see Table 11). In anotherembodiment, Compound 1 Form II (or a pharmaceutically acceptable saltthereof) is formulated as a tablet of formulation 2 (see Table 11). Inyet another embodiment, Compound 1 Form II (or a pharmaceuticallyacceptable salt thereof) is formulated as a suspension of formulation 3(see Table 12). In another embodiment, Compound 1 Form II (or apharmaceutically acceptable salt thereof) is formulated as a suspensionof formulation 4 (see Table 12).

A composition comprising morphic Form II of the compound according tothe present disclosure can be produced by a purification processcomprising recrystallizing a preparation of phosphonic acid,[[(S)-2-(4-amino-2-oxo-1(2H)-pyrimidinyl)-1-(hydroxymethyl)ethoxy]methyl]mono[3-(hexadecyloxy)propyl]ester (Compound 1) frommethanol.

In some preferred embodiments, the Compound 1 produced by the processset forth herein is purified by a series of five distinctrecrystallizations. For instance, Compound 1 can first be purified bythree distinct, sequential recrystallizations from methanol. Then,Compound 1 can be recrystallized from n-heptane and methanol. In somepreferred embodiments, the methanol recrystallizations remove adifferent set of impurities than the n-heptane/methanolrecrystallization. Finally, Compound 1 can be purified again frommethanol. In some preferred embodiments, the final recrystallizationfrom methanol is carried out with seeding with an amount of morphic FormII and proceeds with a slow, controlled cooling that ensures theformation of morphic Form II.

The present disclosure also provides a method for synthesizing acomposition comprising the morphic Form II of Compound 1. The method ofsynthesis of a composition comprising the morphic Form II of the presentdisclosure involves the following steps:

-   -   a. heating a mixture of        (S)—N¹-[(2-hydroxy-3-triphenylmethoxy)propyl]cytosine (Compound        2), P-[[[4-methylphenyl)sulfonyl]oxy]methyl]-,        mono[3-(hexadecyloxy)propyl]ester, sodium salt (Compound 4),        magnesium tert-butoxide, and dimethylformamide (DMF);    -   b. cooling and adding isopropyl acetate;    -   c. washing sequentially with HCl solution and NaCl solution;    -   d. diluting the concentrate with methanol and forming a mixture        containing phosphonic acid,        [[(S)-2-(4-amino-2-oxo-1(2H)-pyrimidinyl)-1-(hydroxymethyl)-2-(triphenylmethoxy)ethyl]methyl]mono[3-(hexadecyloxy)propyl]ester        (Compound 3);    -   e. diluting the concentrate containing Compound 3 in methanol;    -   f. adding HCl gas and maintaining temperature below about 20°        C.;    -   g. filtering to remove impurities and preparing slurry in        acetone;    -   h. filtering the slurry and washing with acetone;    -   i. recrystallizing from methanol;    -   j. recrystallizing a second time from methanol;    -   k. recrystallizing a third time from methanol;    -   l. recrystallizing from n-heptane and methanol;    -   m. dissolving crude product in methanol;    -   n. adding to the solution a seed stock of phosphonic acid,        [[(S)-2-(4-amino-2-oxo-1(2H)-pyrimidinyl)-1-(hydroxymethyl)        ethoxy]methyl]mono[3-(hexadecyloxy)propyl]ester (Compound 1) and        stirring;    -   o. cooling, filtering, and washing the solution with methanol        and drying;        thereby synthesizing morphic Form II of phosphonic acid,        [[(S)-2-(4-amino-2-oxo-1(2H)-pyrimidinyl)-1-(hydroxymethyl)        ethoxy]methyl]mono[3-(hexadecyloxy)propyl]ester (Compound 1).

The present disclosure provides Form II synthesized is more than orequal to about 91% wt/wt, more than or equal to about 95% wt/wt, or morethan or equal to about 99% wt/wt pure.

The present disclosure also provides a method for synthesizing acomposition comprising the morphic Form II of Compound 1. The method ofsynthesis of a composition comprising the morphic Form II of the presentdisclosure involves the following steps:

heating a mixture of(S)—N¹-[(2-hydroxy-3-triphenylmethoxy)propyl]cytosine (Compound 2),P-[[[4-methylphenyl)sulfonyl]oxy]methyl]-,mono[3-(hexadecyloxy)propyl]ester, sodium salt (Compound 4), magnesiumtert-butoxide, and dimethylformamide (DMF) at between about 75-85° C.for about 3 hours;

-   -   a. cooling the mixture to about 25-35° C. and adding isopropyl        acetate;    -   b. further cooling the solution to about 15-25° C. and washing        sequentially with HCl solution and NaCl solution;    -   c. removing isopropyl acetate by vacuum distilling the organic        phase thereby forming a concentrate;    -   d. diluting the concentrate with methanol and further removing        isopropyl acetate thereby re-concentrating and forming a mixture        containing phosphonic acid,        [[(S)-2-(4-amino-2-oxo-1(2H)-pyrimidinyl)-1-(hydroxymethyl)-2-(triphenylmethoxy)ethyl]methyl]mono[3-(hexadecyloxy)propyl]ester        (Compound 3);    -   e. diluting the concentrate containing Compound 3 in methanol;    -   f. adding HCl gas at a rate that maintains the temperature        between about −5 and 15° C.;    -   g. maintaining the reaction about 10-20° C. for 2 hours before        filtering to remove solid impurities;    -   h. diluting the filtrate with water and adjusting pH to about        2.3-2.7 with NaOH;    -   i. filtering the solids and washing the solids with water before        preparing a slurry in acetone at about 35-45° C. for about 1        hour;    -   j. filtering the slurry and washing with acetone;    -   k. drying the acetone washed crude product at a temperature of        equal to or less than about 40° C. for about 12 hours;    -   l. heating the crude product at about 60-70° C. in methanol    -   m. polish filtering, cooling to about 58-62° C., stirring for        one hour, cooling to about 48-52° C. for about six hours, then        to about 17-23° C. for two hours, filtering, and then washing        with methanol;    -   n. repeating steps 1-m twice or more;    -   o. heating the product in methanol at about 64° C. and slowly        adding n-heptane over 40 min while keeping the temperature above        50° C.;    -   p. holding at a temperature about 55° C. for 30 min and cooling        to 40° C. over a 6 h period;    -   q. stirring at 40° C. for 2 h, then cooling to 20° C. over six        hours;    -   r. stirring for 2 h at 20° C.    -   s. filtering and washing with n-heptane and methanol and drying        under vacuum;    -   t. dissolving solids in methanol before cooling to about        59-61° C. and then stirring for about 20 minutes;    -   u. adding to the solution a seed stock of phosphonic acid,        [[(S)-2-(4-amino-2-oxo-1(2H)-pyrimidinyl)-1-(hydroxymethyl)        ethoxy]methyl]mono[3-(hexadecyloxy)propyl]ester (Compound 1) and        stirring for two hours;    -   v. cooling the solution to about 47-53° C. by stirring for about        eight hours, and then stirring for about two hours;    -   w. cooling the stirred solution further to about 17-23° C. over        about six hours, and further stirring for about two hours;    -   x. filtering and washing the solution with methanol;    -   y. drying at a temperature of equal to or less than about 40° C.        for about twenty-four hours; and    -   thereby synthesizing morphic Form II of phosphonic acid,        [[(S)-2-(4-amino-2-oxo-1(2H)-pyrimidinyl)-1-(hydroxymethyl)        ethoxy]methyl]mono[3-(hexadecyloxy)propyl]ester (Compound 1).

The present disclosure provides a method of preparing Form II ofCompound 1, comprising the step of combining(S)—N¹-[(2-hydroxy-3-triphenylmethoxy)propyl]cytosine (Compound 2),P-[[[4-methylphenyl)sulfonyl]oxy]methyl]-,mono[3-(hexadecyloxy)propyl]ester, sodium salt (Compound 4) withmagnesium tert-butoxide, and dimethylformamide (DMF).

The present disclosure provides a method for synthesizing a crystallinemorphic Form II of Compound 1, by performing a crystallization step. Forinstance, a solution (e.g., a methanol solution) comprising Compound 1can be seeded with about 0.5%, about 3%, or about 7% of seed of thephosphonic acid (e.g., the seed can be Form I or Form II of Compound 1).The solution can then be allowed to crystallize. In some embodiments,the crystalline morphic form II of Compound 1 is crystallized with slowstirring or agitation during the crystallization process. In oneembodiment, the rate of stirring affects the type of morphic formproduced. In another embodiment, the rate of stirring does not affectthe type of morphic form produced. The embodiments provide morphic FormII crystallization by a process comprising methanol. The embodimentsfurther provide crystallization process for forming morphic Form II byseeding with Form II or Form I of the phosphonic acid. Additionally, insome embodiments, morphic Form II is provided by cooling the methanolslowly. For instance, in some preferred embodiments, Compound 1 can bedissolved in methanol at about 65° C. (e.g., about reflux temperature)and held at about 65° C. for 1 hour then cooled to 61° C. before seedingwith morphic Form II. The contents can be stirred at 60° C. for 1 hourand cooled to 50° C. over a period of eight hours. The contents can beheld at 50° C. for two hours, and then further cooled from 50° C. to 20°C. for at least six hours (e.g., overnight) and stirred at 20° C. for atleast two hours before filtering. In some preferred embodiments, theslow cooling protocol can help ensure the formation of Form II.

In one embodiment, the present disclosure provides production of Form IIwith starting material about 100 Kg without seeding. The process ofproduction of Form II without seeding involves quick cool-down methanolrecrystallization, which results in smaller particle size compared tothe particle size of Form II when the process includes seeding and slowcool-down.

In some embodiments, a composition comprising morphic Form IIsynthesized following the method of the present disclosure issubstantially free of impurities. In some embodiments, a compositioncomprising morphic Form II synthesized following the method of thepresent embodiment is more than or equal to about 99% wt/wt pure. Insome preferred embodiments, the substantial (e.g., about 100% pure)purity is the result of a series of recrystallizations that can remove ahost of impurities. In some embodiments, although any particularrecrystallization technique can remove impurities, certainrecrystallizations can be more effective at removing certain specificimpurities compared with other recrystallizations. For instance, in someembodiments, three initial recrystallizations from methanol removecertain impurities. Next, in some embodiments, a recrystallization fromn-heptane and methanol can remove additional (e.g., different)impurities remaining in Compound 1 that were not fully removed by therecrystallizations from methanol alone. A final recrystallization frommethanol with seeding and a slow cool-down can remove additional traceimpurities and produce morphic Form II of Compound 1.

In some embodiments, a composition comprising morphic Form IIsynthesized by the method of the present disclosure can be a hydrate. Insome embodiments, a composition comprising the morphic Form II of thecompound of the present disclosure is not a hydrate. In someembodiments, a composition comprising the morphic Form II synthesized bythe method of the present disclosure is substantially free of thehydrate form. In some embodiments, a composition comprising the morphicform II partially hydrated or a partial hydrate.

In some embodiments, a composition comprising morphic Form II of thepresent disclosure partially converts to a hydrate form after beingexposed to about 43% RH for about 12 days. In some embodiments, acomposition comprising The morphic Form II of the present embodimentshows a minor endotherm at about 41-43° C. (peak max) followed byoverlapping major endotherms at about 90 and about 95° C. (peak max) ina DSC thermogram. The final endotherm of a composition comprising themorphic Form II of the present embodiment has an onset at about 196° C.

The present disclosure provides a method of synthesis of a morphic FormII of a compound having Formula II or III characterized by an X-raydiffraction pattern with two or more (e.g., three or more, four or more,five or more, six or more, seven or more, eight or more, nine or more,or ten or more) peaks expressed in degrees 2θ (±0.2) selected from 2.81,5.63, 11.30, 12.05, 13.22, 13.45, 13.81, 14.32, 14.92, 15.64, 16.25,16.41, 17.00, 17.67, 17.87, 18.15, 18.35, 18.50, 19.00, 19.57, 19.85,20.22, 20.96, 21.06, 21.89, 22.76, 23.70, 23.95, 24.32, 24.70, 25.54,26.12, 26.52, 26.81, 27.07, 27.48, 27.71, 29.11, 29.36, and 29.61.

The present disclosure further provides a method of synthesis of amorphic Form II of a compound having Formula II or III characterized byan X-ray diffraction pattern substantially similar to that set forth inFIG. 1, FIG. 7, FIG. 13, FIG. 14, FIG. 20, FIG. 21, or FIG. 25.

Some embodiments of the present disclosure provides a method ofsynthesis of a morphic Form II of a compound having Formula II or IIIcharacterized by a DSC Thermogram substantially similar to that setforth in FIG. 4, FIG. 15, or FIG. 16.

The present disclosure provides a Form II of a compound of Formula II orIII in a stable crystalline form.

The present disclosure provides a method of synthesizing(S)—N¹-[(2-hydroxy-3-triphenylmethoxy)propyl]cytosine (Compound 2),including the following steps:

-   -   a. heating a mixture of (S)-trityl glycidyl ether, cytosine,        potassium carbonate, and N,N-dimethylformamide (DMF);    -   b. cooling the reaction mixture and quenching with toluene;    -   c. cooling the resulting slurry of step b., filtering, washing        with toluene;    -   d. slurrying the solids in toluene, filtering, and then washing        with acetone;    -   e. triturating the solids in water/acetone, filtering, and        washing with and suspending in acetone;    -   f. optionally repeating steps d-e to remove residual cytosine        and process-related impurities; and    -   g. drying the filter cake in vacuo, thereby yielding        (S)—N¹-[(2-hydroxy-3-triphenylmethoxy)propyl]cytosine (Compound        2).

In one embodiment, HPLC (AUC) purity of the synthesized compound isequal to or greater than 91% wt/wt, equal to or greater than 91% wt/wt,or equal to or greater than 99% wt/wt.

The present disclosure also provides a method of synthesizing(S)—N¹-[(2-hydroxy-3-triphenylmethoxy)propyl]cytosine (Compound 2),including the following steps:

-   -   a. heating a mixture of (S)-trityl glycidyl ether, cytosine,        potassium carbonate, and N,N-dimethylformamide (DMF) at about        85-95° C. for about 9 hours;    -   b. cooling the reaction mixture of step a. to about 66-70° C.        and quenching with toluene;    -   c. cooling the resulting slurry of step b. to about −10 to 5°        C., filtering, washing with toluene;    -   d. slurrying the solids in toluene (168.8 kg) at about 15-25°        C., filtering, and then washing with acetone;    -   e. triturating the solids in water/acetone (90.0 kg/54.0 kg) at        about 17-22° C., filtering, and washing with acetone (36.0 kg);    -   f. suspending filter cake of the filtrate in acetone (178.9 kg)        and heated at approximately about 35-45° C. for about 3 hours,        filtered, and washed with acetone (36.0 kg).    -   g. The washes and triturations are optionally repeated as needed        to remove residual cytosine and process-related impurities. The        cake is dried in vacuo at a temperature equal to or less than        about 40° C. for about 12 hours to yield about 45.0 kg (about        65.0%) of Compound 2. HPLC (AUC) purity of the synthesized        compound is equal to or greater than 99%.

A number of unique features and advantages set forth in the presentdisclosure will become apparent to one of skill in the art and areelaborated in the Detailed Description below. A first advantage of thepresent invention is increased purity of the parent compound,Compound 1. It is readily understood in the chemical and pharmaceuticalarts that purity is of utmost importance when characterizing compoundsand dosing them for use as pharmaceutical agents. In some preferredembodiments, the current technology provides for five distinctrecrystallizations of Compound 1. In some preferred embodiments, thefirst three recrystallizations are from methanol and are used to removecertain impurities (e.g., Compounds A and B, below) from Compound 1. Insome preferred embodiments, a fourth recrystallization (e.g., ofCompound 1) from n-heptane and methanol is employed to remove otherimpurities (e.g., Compounds C and D, below) that remain after theinitial three methanol recrystallizations. Accordingly, the presenttechnology provides a system of using distinct and orthogonalpurification methods to effectively remove impurities in Compound 1 andto arrive at a composition of Compound 1 that is substantially(e.g., >99% or >99.9%) pure.

A second advantage of the present invention is the ability to generate aspecific polymorph of Compound 1 for use in pharmaceutical manufacturing(e.g., commercial pharmaceutical manufacturing). In some preferredembodiments, the fifth recrystallization (e.g., from methanol with aslow cooling and seeding with Form II) can produce Form II reliably andconsistently. A skilled artisan will readily recognize that a givencompound (e.g., Compound 1) can exist as a variety of differentpolymorphs. These polymorphs can have significantly different physicalproperties such as density, solubility, and even chemical reactivity.The heterogeneity between different polymorphs of the same compound canconfuse efforts to reliably provide accurate and precise dosages of agiven pharmaceutical agent (e.g., Compound 1). For instance, if twodifferent polymorphs of the same compound have different densities, itcan be difficult to formulate a pharmaceutical composition comprisingthat compound that reliably has the same amount, on a molar basis, ofthe compound itself. Similarly, if different polymorphs have differentsolubility profiles, they can act differently in the body (e.g.,different polymorphs may dissolve faster or slower in the blood). Thiscan complicate efforts to reliably provide, for example, animmediate-release or an extended release dosage form. In other words,polymorphism can be responsible for inconsistencies encountered in theperformance of different pharmaceutical agents. See, e.g., Caira, M. R.Crystalline Polymorphism in Organic Compounds. Accordingly, a polymorphof a given compound (e.g., Compound 1) can have unique properties thatare not inherent in the compound (e.g., Compound 1) itself.

Additionally, it is not routine practice to make a specific polymorph.Indeed, in some cases it can be unpredictable and/or unreliable toarrive at a specific polymorph and the process for producing them canfail to generate them consistently or reliably. See Polymorphism inPharmaceutical Solids, 2nd Ed., Drugs and the Pharmaceutical Sciences(2009), Informa Healthcare USA, Inc., New York, N.Y., Chapter 3, page52, Introduction and Chapter 4, page 77, first full paragraph and page87, first full paragraph.

The present invention provides ways to arrive at a specific polymorph(i.e., Form II of Compound 1) consistently and reliably. In somepreferred embodiments, Form II is generated by seeding the finalrecrystallization from methanol with an appropriate amount of a seedcrystal of Form II. Also, in some preferred embodiments, therecrystallization proceeds with a slow cooling ramp to ensure theformation of morphic Form II of compound 1. In some preferredembodiments, the combination of seeding with morphic Form II and a slowcool-down procedure can reliably and predictably ensure the formation ofmorphic form II from the final methanol recrystallization.

In some embodiments, the compound of Formula II or III (e.g.,Compound 1) can be in amorphous form.

In one or more aspects, the present invention provides morphic Form IIof phosphonic acid,[[(S)-2-(4-amino-2-oxo-1(2H)-pyrimidinyl)-1-(hydroxymethyl)ethoxy]methyl]mono[3-(hexadecyloxy)propyl]ester (Compound 1).

Additional features and advantages of the current technology such asimproved yield of the process and a reduction in time for synthesizingCompound 1 will also be apparent to a skilled artisan.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. In the case of conflict, thepresent specification, including definitions, will control. In thespecification, the singular forms also include the plural unless thecontext clearly dictates otherwise. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference. The references cited herein are not admitted to be prior artto the claimed invention. In addition, the materials, methods, andexamples are illustrative only and are not intended to be limiting.

BRIEF DESCRIPTION OF THE FIGURES

Other features and advantages of the invention will be apparent from thefollowing detailed description and claims.

FIG. 1 XRPD scatter for a sample of Compound 1, Form II (sample 1).

FIG. 2 shows indexing results of Compound 1 Form II for XRPD collectedwith Cu-Kα radiation (sample 1).

FIG. 3 shows indexing results of Compound 1 Form H for XRPD collectedwith Cu-Kα radiation.

FIG. 4 shows DSC thermogram of Compound 1, Form II (sample 1).

FIG. 5 shows particle size distribution of Compound 1, Form II, withvolume weighted mean as 25.663.

FIG. 6 shows ¹H NMR spectrum of Compound 1, Form II (sample 1).

FIG. 7 shows XRPD scatter for a sample of Compound 1, Form II (sample2).

FIG. 8 shows DSC thermogram of Compound 1, Form II (sample 2).

FIG. 9 shows comparison of XRPD patterns of Compound 1, Form I, II(sample 1), and H.

FIG. 10 shows XRPD overlay of Compound 1 drug product samples (Tablet 1(upper plot) and Tablet 2 (lower plot)).

FIG. 11 shows XRPD overlay of Compound 1 drug product samples with FormII, Form I (Tablet 1), and Form H.

FIG. 12 shows solubility curve and MSZ of Compound 1, Form II (sample 1)in methanol.

FIG. 13 shows XRPD of Form II, Tablet 1.

FIG. 14 shows XRPD of Form II, Tablet 2.

FIG. 15 shows DSC thermogram of Form II, Tablet 1. Endotherms are shownat about 90, 93, and 165° C.

FIG. 16 shows DSC thermogram of Form II, Tablet 2. Endotherms are shownat about 89, 94, and 165° C.

FIG. 17 shows thermal analysis of Form H (DSC Thermogram).

FIG. 18 shows XRPD overlay (˜15-30 2-Theta) of two Form H samples ofCompound 1 with peak shifting.

FIG. 19 shows a comparison of particle size distribution overlay ofCompound 1 Form II samples produced by recrystallized from methanol andrapid cooling (cross) with samples 3 (circle) and 4 (triangle) producedwith seeding and slow cooling.

FIG. 20 shows XRPD scatter for a sample of Compound 1, Form II (sample3).

FIG. 21 shows a second XRPD scatter for a sample of Compound 1, Form II(sample 3).

FIG. 22 shows DSC thermogram of Compound 1, Form II (sample 3).

FIG. 23 shows a close-up DSC thermogram of Compound 1, Form II (sample3).

FIG. 24 shows another DSC thermogram of Compound 1, Form II (sample 3).

FIG. 25 shows a comparison XRPD overlay of Compound 1, Form II (sample3); Compound 1, Form II (sample 1); and Compound 1, Form H.

FIG. 26 shows a comparison DSC thermogram overlay of Compound 1 Form II(sample 3); Compound 1, Form II and Compound 1, Form H.

FIG. 27 shows a chromatogram of Compound 1 after two fast cool-downmethanol recrystallizations. The chromatogram shows that Compound 1 is98.5% pure.

FIG. 28 shows a chromatogram of Compound 1, Form II after threeslow-cool down methanol recrystallizations with form control. Thechromatogram shows that Compound 1 is 99.5% pure.

FIG. 29 shows a chromatogram of Compound 1, Form II after oneheptane/methanol recrystallization followed by one methanol slowcool-down recrystallization for form control. The chromatogram showsthat Compound 1 is 100% pure.

FIG. 30 shows a chromatogram of Compound 1, Form II after aheptane/methanol recrystallization followed by three methanolrecrystallizations. The chromatogram shows that Compound 1 is 100% pure.

DETAILED DESCRIPTION OF THE INVENTION

The solid form (e.g., crystal state) of a compound may be important whenthe compound is used for pharmaceutical purposes. Compared with anamorphous solid, the solid physical properties of a crystalline compoundmay change from one solid form to another, which may impact itssuitability for pharmaceutical use. In addition, different solid formsof a crystalline compound may incorporate different types and/ordifferent amounts of impurities. Different solid forms of a compound mayalso have different chemical stability upon exposure to heat, lightand/or moisture (e.g., atmospheric moisture) over a period of time, ordifferent rates of dissolution. There remains a need for solidcrystalline forms of phosphonic acid,[[(S)-2-(4-amino-2-oxo-1(2H)-pyrimidinyl)-1-(hydroxymethyl)ethoxy]methyl]mono[3-(hexadecyloxy)propyl]ester (“Compound 1”) that arenot hygroscopic, and that exhibit improved chemical stability for use indrug substance and drug product development.

The ability of a substance to exist in more than one crystal form isdefined as polymorphism; the different crystal forms of a particularsubstance are referred to as “polymorphs” of one another. In general,polymorphism is affected by the ability of a molecule of a substance (orits salt or hydrate) to change its conformation or to form differentintermolecular or intra-molecular interactions (e.g., different hydrogenbond configurations), which is reflected in different atomicarrangements in the crystal lattices of different polymorphs. Incontrast, the overall external form of a substance is known as“morphology,” which refers to the external shape of the crystal and theplanes present, without reference to the internal structure. Aparticular crystalline polymorph can display different morphology basedon different conditions, such as, for example, growth rate, stirring,and the presence of impurities.

The different polymorphs of a substance may possess different energiesof the crystal lattice and, thus, in solid state they can show differentphysical properties such as form, density, melting point, color,stability, solubility, dissolution rate, etc., which can, in turn,effect the stability, dissolution rate and/or bioavailability of a givenpolymorph and its suitability for use as a pharmaceutical and inpharmaceutical compositions. See, e.g., Polymorphism in PharmaceuticalSolids, 2nd Ed., Drugs and the Pharmaceutical Sciences (2009), InformaHealthcare USA, Inc., New York, N.Y., Chapter 3, page 52, Introductionand Chapter 4, page 77, first full paragraph and page 87, first fullparagraph and Caira M. R., Crystalline Polymorphism of OrganicCompounds.

U.S. Pat. No. 8,569,321 discloses crystalline physical form of Compound1, which is designated as Form A. The present invention includes newpolymorphic form of Compound 1, which is a stable polymorph, and methodfor preparation thereof.

Compound 1 has been produced at about 100 kg scale utilizing a rapidcooling crystallization process from methanol. The process of productionof Form II without seeding involves quick cool-down methanolrecrystallization, which results in smaller particle size compared tothe particle size of Form II when the process includes seeding and slowcool-down steps.

The present disclosure relates to (i) characterization of selectedCompound 1 morphic forms to evaluate solid form and particle differencesamong the morphic forms, (ii) solid form screening focused on theprocess solvents to evaluate the propensity of Compound 1 to producedifferent crystal forms, and (iii) an improved crystallization processwhich is more controlled and reproducible.

The present disclosure provides methods of synthesis for substitutedphosphonic acid esters. In certain aspects, the invention providesmethods for the preparation of compounds having the structure of FormulaI:

wherein:

R¹ is unsubstituted or substituted C₁-C₆ alkoxy-, or unsubstituted orsubstituted C₁-C₃₀ alkoxy-C₁-C₆-alkoxy-; or an enantiomer, diastereomer,racemate or a mixture thereof

In another embodiment, R¹ is C₁₀-C₃₀ alkoxy-C₂-C₄-alkoxy-.

In another embodiment, R¹ is hexadecyloxypropyloxy-.

Provided herein are novel crystalline forms of Compound 1.

In one embodiment, the present disclosure provides methods for thepreparation of compounds having the structure of Formula II:

or enantiomer, diastereomer, racemate or a mixture thereof, or apharmaceutically acceptable salts thereof.

In one embodiment, the present disclosure provides methods for thepreparation of compounds having the structure of Formula III:

or a pharmaceutically acceptable salt thereof.

Morphic Forms I, II, & H

Described herein are polymorphic Forms I, II and H of Compound 1 (alsoreferred to herein respectively as “Polymorph I”, “Polymorph II” and“Polymorph H”).

The Form II of Compound 1 can be produced in a highly crystalline form,which is useful in the preparation of pharmaceutical formulations, andwill improve general handling, manipulation, and storage of the drugcompound. In an embodiment, the crystalline form of the Form II of acompound having Formula II or III is in a form referred to as “PolymorphII,” “morphic Form II” or “Form II.” (“Polymorph,” “morphic Form,” or“Form” is used interchangeably throughout the present disclosure.) Asdescribed herein, Polymorph II or Form II exhibits physical propertiesthat can be exploited in order to obtain new pharmacological properties,and that may be utilized in drug substance and drug product development.

Polymorph II (or “morphic Form II” or “Form II”) has a number ofadvantageous physical properties related to its free acid form, andrelated to other polymorphs. Form II has low hygroscopicity compared toother polymorphs (e.g., Form I and/or Form H) of the compound of FormulaII or III. Form II has low hygroscopicity compared to another polymorphform of Compound 1 (e.g., Form I and/or Form H). For consistency withdrug formulation (e.g., tableting), it is generally required that thepolymorphic form of the active pharmaceutical ingredient (API) compoundbe minimally hygroscopic. Drug forms that are highly hygroscopic mayalso be unstable, as the drug form's dissolution rate (and otherphysico-chemical properties) may change as it is stored in settings withvarying humidity. Also, hygroscopicity can impact large-scale handlingand manufacturing of a compound, as it can be difficult to determine thetrue weight of a hygroscopic active agent when preparing apharmaceutical composition comprising that agent. For example, in largescale tableting or other medicinal formulating preparations, highlyhygroscopic compounds can result in batch manufacturing inconsistencycreating clinical and/or prescribing difficulties. Form II may have lowhygroscopicity compared to other polymorphs (e.g., Form I and/or Form H)of the compound of Formula II or III. As such, morphic Form II is storedover appreciable periods or conditions (e.g., low relative humidityconditions), without substantial or any detrimental formulating changes.

In one embodiment, the present disclosure provides a morphic Form II ofCompound 1. The present disclosure provides a morphic Form II which isanhydrous.

In some embodiments, Compound 1 is substantially free of impurities. Insome embodiments, the purity of Compound 1 or a pharmaceuticallyacceptable salt thereof is equal to or greater than 92% (e.g., ≧92%,≧93%, ≧94%, ≧95%, ≧96%, ≧97%, ≧98%, ≧99%, or ≧99.5%). In yet otherembodiments, Compound 1 or a pharmaceutically acceptable salt thereofhas a purity of equal to or greater than 91% (e.g., ≧91%, ≧92%, ≧93%,≧94%, ≧95%, ≧96%, ≧97%, ≧98%, ≧99%, or ≧99.5%). In one embodiment, thepurity of Compound 1 or a pharmaceutically acceptable salt thereof isabout 99%. In one embodiment, the Compound 1 polymorph is a hydrate. Inanother embodiment, the Compound 1 polymorph is not a hydrate. In yetanother embodiment, the compound is a solvate, e.g., a methanol solvate,an ethanol solvate, or an isopropanol solvate.

In some embodiments, the purity of the compound of Formula I, II, and/orIII (or a pharmaceutically acceptable salt thereof) is equal to orgreater than 92% (e.g., ≧92%, ≧93%, ≧94%, ≧95%, ≧96%, ≧97%, ≧98%, ≧99%,or ≧99.5%). In other embodiments, the compound is in Form II or apharmaceutically acceptable salt thereof. In yet other embodiments,Compound 1 is Form II and has a purity of equal to or greater than 91%(e.g., ≧91%, ≧92%, ≧93%, ≧94%, ≧95%, ≧96%, ≧97%, ≧98%, ≧99%, or ≧99.5%).In one embodiment, the purity of the compound of Formula I, II, and/orIII (or a pharmaceutically acceptable salt thereof) is about 99%. In oneembodiment, the compound of Formula I, II, or III (or a pharmaceuticallyacceptable salt thereof) is obtained from recrystallizing a crudecompound from a suitable recrystallizing solvent described herein.

The present disclosure provides synthesizing Compound 1 or apharmaceutically acceptable salt thereof by varying the temperature andmethanol:water ratio. The present disclosure provides synthesizing FormII of the compound of Formula II or III (or a pharmaceuticallyacceptable salt thereof) by varying the temperature and methanol:waterratio. In alternative temperature and methanol:water ratios, hydrateform (e.g., Form H) or a mixture of hydrate form (e.g., Form H) and ananhydrate form (e.g., Form II) is formed.

In some embodiments, an anhydrous form (e.g., Form II) of the compoundof Formula II and/or III (or a pharmaceutically acceptable salt thereof)is obtained during crystallization process by setting up slurries indifferent methanol-water concentrations. In one embodiment, fromsub-ambient slurries (i.e., slurries prepared in the temperature of thesurroundings), methanol:water ratio of about 98:2 or 99:1 recoveranhydrous morphic form (e.g., Form II). In contrast, in anotherembodiment, methanol:water ratio of about 97:3 or greater recovershydrate morphic Form (e.g., Form H) either in mixture with Form II or asa pure form of Form H. The present disclosure provides that waterconcentration of equal to or no more than between about 1 and 3% isrequired to synthesize an anhydrous Form (e.g., Form II) of the compoundof Formula II or III (or a pharmaceutically acceptable salt thereof).The water content of such embodiments vary depending on the watercontent in the methanol, and is more than about 1 to 3% for synthesizingan anhydrous Form (e.g., Form II) of the compound of Formula II or III(or a pharmaceutically acceptable salt thereof).

In another embodiment, from slurries at room-temperature, an anhydrousmorphic form (e.g., Form II) of the compound of Formula II or III (or apharmaceutically acceptable salt thereof) is obtained in the presence ofequal to or less than 5% (e.g., ≦5—4.9%, ≦4.9—4.8%, ≦4.8—4.7%,≦4.7—4.6%, ≦4.6—4.5%, ≦4.5—4.4%, ≦4.4—4.3%, ≦4.3—4.2%, ≦4.2—4.1%,4.1-4.0%, ≦4.0—3.0%, or ≦3.0—2.0%) water in the slurry. In oneembodiment, from slurries at room-temperature, the critical waterconcentration to obtain an anhydrous morphic form (e.g., Form II) of thecompound of Formula II or III (or a pharmaceutically acceptable saltthereof) is equal to or less than 5%.

In yet another embodiment, from slurries at a temperature higher thanthe room temperature (e.g., equal to or more than 30° C.), an anhydrousmorphic form (e.g., Form II) of the compound of Formula II or III isobtained in the presence of equal to or less than 10% (e.g., ≦10—9%,≦9—8%, ≦8—7%, ≦7—6%, ≦6—5%, ≦5—4%, ≦4—3%, or ≦3—2%) water in the slurry.In one embodiment, from slurries at about 45° C., the critical waterconcentration to obtain an anhydrous morphic form (e.g., Form II) of thecompound of Formula II or III (or a pharmaceutically acceptable saltthereof) is equal to or less than 10%.

In some embodiments, a composition comprising the morphic Form II of thecompound of Formula II or III (or a pharmaceutically acceptable saltthereof) is substantially free of Form I and/or Form H. For example, acomposition comprising the morphic Form II of the present disclosurecomprises equal to or less than 10% Form I and/or Form H. In someembodiments, the Form II composition comprises equal to or less than 9%,equal to or less than 8%, equal to or less than 7%, equal to or lessthan 6%, equal to or less than 5%, equal to or less than 4%, equal to orless than 3%, equal to or less than 2%, equal to or less than 1%, equalto or less than 0.9%, equal to or less than 0.8%, equal to or less than0.7%, equal to or less than 0.6%, equal to or less than 0.5%, equal toor less than 0.4%, equal to or less than 0.3%, equal to or less than0.2%, equal to or less than 0.1%, equal to or less than 0.05%, equal toor less than 0.01%, or equal to or less than 0.001% Form I and/or FormH.

In some embodiments, a composition comprising morphic Form II of thepresent disclosure converts to a hydrate Form (e.g., Form H) if storedunder relative humidity (RH) of 40% or more. In some embodiments, acomposition comprising morphic Form II of the present disclosurepartially converts to morphic Form H if stored under relative humidityof equal to or more than 40% for several days. In some embodiments, acomposition comprising the morphic Form II partially converts to ahydrate form (e.g., Form H) after exposure to about 43% relativehumidity (RH) for about 12 days. A complete conversion of a compositioncomprising the morphic Form II of the present disclosure occurs at aboutequal to or more than 80% RH, about equal to or more than 81% RH, aboutequal to or more than 82% RH, about equal to or more than 83% RH, aboutequal to or more than 84% RH, about equal to or more than 85% RH, aboutequal to or more than 86% RH, about equal to or more than 87% RH, aboutequal to or more than 88% RH, about equal to or more than 89% RH overbetween 5-20 days (e.g., 5 days, 6 days, 7 days, 8 days, 9 days, 10days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18days, 19 days, 20 days).

The present disclosure provides that exposure of Form II to different RHstressing conditions from about 43 to about 85% RH for about 12 daysresults in minor conversion from Form II to Form H. In one embodiment,at about 85%, RH complete conversion of Form II to Form H occurs. SeeTable 1. The present disclosure provides that exposure of Form II tohigher humidity is sufficient for conversion to the hydrate form.

TABLE 1 Conversion of Form II to Form H Temp Solvent^(a) (° C.)Observation Results Methanol: water (99:1) ~2-8 Opaque aggregates andForm II fines with no distinct Methanol: water (97:3) ~2-8 Opaqueaggregates and Form H fines with no distinct Methanol: water (95:5) ~2-8Opaque aggregates and Form H fines with no distinct Methanol: water(93:7) ~2-8 Opaque aggregates and Form H fines with no distinctMethanol: water (97:3) RT Opaque aggregates and Form II + fines with nodistinct Minor Form H Methanol: water (95:5) RT Opaque aggregates andForm H fines with no distinct Methanol: water (93:7) RT Opaqueaggregates and Form H fines with no distinct Methanol: water (90:10) RTOpaque aggregates and Form H fines with no distinct Methanol: water(95:5) ~44-45 Opaque aggregates and Form II + fines with no distinctMinor Form H Methanol: water (90:10) ~44-45 Opaque aggregates and Form Hfines with no distinct Methanol: water (85:15) ~44-45 Opaque aggregatesand Form H fines with no distinct Methanol: water (80:20) ~44-45 Opaqueaggregates and Form H fines with no distinct ^(a)Slurry experiments wereprepared with Form II (Compound 1 (Compound 1)) and seeded with a smallamount of Form H. Samples were left to stir for 7-9 days. Solubility

The present disclosure provides morphic Form II with low solubility inmethanol. The solubility may be estimated based on the total volume ofsolvent needed to provide complete dissolution. The actual solubilitymay be greater than the value calculated due to the incremental additionof solvent and kinetics of dissolution of the material. The solubilityis expressed as “less than” if dissolution did not occur during theexperiment, or “more than” if dissolution occurred after the addition ofthe first aliquot.

TABLE 2 Solubility Definitions Term Definition Low solubility <1 mg/mLLimited solubility 1-20 mg/mL Intermediate solubility 20-100 mg/mL Goodsolubility 100-200 mg/mL High solubility >200 mg/mL

In some embodiments, a composition comprising the morphic Form II of thepresent disclosure is equal to or less than about 5 mg/mL soluble (e.g.,≦5—4 mg/mL, ≦4—3 mg/mL, ≦3—2 mg/mL, ≦2—1 mg/mL, or ≦1—0.01 mg/mL) in 1:1methanol:water ratio at room temperature. In one embodiment, acomposition comprising the morphic Form II of the present disclosure isless than about 3 mg/mL soluble in 1:1 methanol:water ratio at roomtemperature. In some embodiments, a composition comprising the morphicForm II of the present disclosure is equal to or less than about 15mg/mL soluble (e.g., ≦15—14 mg/mL, ≦14—13 mg/mL, ≦13—12 mg/mL, ≦12—11mg/mL, ≦11—10 mg/mL, ≦10—9 mg/mL, ≦9—8 mg/mL, ≦8—7 mg/mL, 7-6 mg/mL, or6-5 mg/mL) in 1:1 methanol:water ratio at a temperature higher than roomtemperature (e.g., equal to or more than 30° C.). In one embodiment, acomposition comprising the morphic Form II of the present disclosure isless than about 14 mg/mL soluble at about 63° C.

TABLE 3 Estimated Solubility of Form II Solvent Temperature (° C.)Solubility (mg/mL) ^(a) Methanol: Water 1:1 RT <3 ~63 <14 ^(a)Solubility was estimated based on the total solvent used to give asolution; actual solubility may be greater because of the volume of thesolvent portions utilized or a slow rate of dissolution. Solubility isrounded to the nearest mg/mL.

TABLE 4 Methanol Solubility and Metastable Zone (MSZ)* Data of Form IIConcentration^(a) Clear Point Cloud Point 1^(b) Cloud Point 2^(c)(mg/mL) (° C.) (° C.) (° C.) 20.3 53.6 37.6 38.2 29.4 56.2 45.1 44.638.9 58.1 45.1 51.1 50.5 60.2 51.0 56.6 64.3 60.5 48.5 58.8 80.4 61.249.7 57.9 95.6 61.9 58.1 59.9 166.6 63.1 58.1 61.1 ^(a)Non-GMPexperiment run in Crystal16 ™ was used as starting material.^(b)Determined from 0.5° C./min cooling rate. ^(c)Determined from 0.03C/min cooling rate. *If the solution mentioned previously is furthercooled, a new solid phase is formed by nucleation at a specific point,which define the metastable zone.

Particle Size of Polymorphic Compound 1

In another embodiment, particle size analysis and Scanning ElectronMicroscopy (SEM) images provide particle characteristics and variationsof characteristics for crystals formed under different crystallizationconditions.

The present disclosure provides an internal structure of Form II of thecompound having Formula II or III (or a pharmaceutically acceptable saltthereof) such that the crystal unit cell dimension and packing aredifferent from internal structures of Form I and Form H. The presentdisclosure also provides a single crystalline form (e.g., Form II) ofthe Formula of II or III (or a pharmaceutically acceptable saltthereof). Form II of the present disclosure is characterized by XRPDindexing, unique DSC characteristics, distinct particle size and form,and lower solubility in methanol.

In one embodiment, Form II forms large agglomerates along with smallerplate-shaped particles. The SEM shows that the Form I of the presentdisclosure have the largest agglomerates (>500 μm) and the surfacesappeared significantly smoother compared to the other two lots. In oneembodiment, the particles do not contain any cemented agglomerates andare composed of larger, thin, plate-shaped particles. The crystalparticles obtained have a bi-modal distribution with a mode of smallparticles at about 6-10 μm and a larger mode at about 60-160 μm. In oneembodiment, the sample has a single mode at about 90 μm with a tail offiner particles. In another embodiment the sample has a large d90(particle diameter at which 90% of the sample is smaller than),consistent with observations from the SEM images.

The present disclosure provides particle sizes of the starting materialfor crystallization; methanol recrystallized material; and comilledmaterial from an about 45 kg recrystallization batch. The startingmaterial has agglomerates (about 100 μm) composed of smaller plates. Themethanol recrystallized forms of the present disclosure has largerprimary particles, some agglomeration without cementation and a singleparticle size mode. The comilled sample is similar to the methanolrecrystallized batch but shows a slightly smaller particle distributionsuggesting only minor particle attrition occurred during the millingstep.

TABLE 5 Particle size distribution of sample 1, 3 and 4 of Compound 1Form II Sample no. d10 (μm)^(a) d50 (μm)^(b) d90 (μm)^(c) file RecordPage Sample 1 19.297 81.603 168.698 613675 1 10, 13 Sample 4 17.41085.440 199.029 613739 2 10, 14 Sample 3 19.896 101.748 274.113 613674 110, 15 ^(a)10% of the total volume of particles is comprised ofparticles no larger than the indicated size in μm. ^(b)50% of the totalvolume of particles is comprised of particles no larger than theindicated size in μm. ^(c)90% of the total volume of particles iscomprised of particles no larger than the indicated size in μm.

Some embodiments provide particles and agglomerates of different sizesand forms. The particle and agglomerate sizes and forms vary dependingon the percentage of seeding. In one embodiment, particles andagglomerates synthesized by a crystallization process seeded with 0.5%of Compound 1 is distinct from the particles and agglomeratessynthesized by a crystallization process seeded with 3% of Compound 1.Compare FIGS. 22 and 23. Particle size analysis of these embodimentsprovide that, due to the larger number of particles/surface areaavailable for crystal growth, the 3% seeded sample has smaller d10(particle diameter at which 10% of the sample is smaller than), d50(particle diameter at which 50% of the sample is smaller than), and d90(particle diameter at which 90% of the sample is smaller than) valuescompared to the 0.5% seeded sample. The samples crystallized using thehydrate e.g., Form H, as the starting material or which contained excesswater during the crystallization, appear to generate samples with alower degree of agglomeration. The higher water content changes thesolubility or induction time and avoids the secondary nucleation andagglomeration.

X-Ray Diffraction

In certain embodiments, Forms I, II, and H of the compound havingFormula II or III are identifiable on the basis of their respectivecharacteristic peaks in an X-ray powder diffraction analysis. X-raypowder diffraction pattern, also referred to as XRPD pattern, is ascientific technique involving the scattering of x-rays by crystalatoms, producing diffraction pattern that yields information about thestructure of the crystal. Internal structure of crystals is accessibleby x-ray diffraction analysis. Polymorphs have different crystal formsbased on their different structures, and physical and chemicalproperties.

Metastable zone width (MSZW) is a critical parameter in thecrystallization process as it reveals the nucleation behavior of thesystem. MSZW is a nucleation kinetic-limited parameter that is highlydependent on process condition. Many factors may influence the value ofMSZW, e.g., rate of cooling, agitation, the presence of foreignparticles and impurities. MSZW decreases as stirrer speed increases;MSZW widens at N>400 rpm; and MSZW widens as cooling rate rises. In oneembodiment, large differences in the metastable zone are observed forboth Form II and Form I between the rapid cooling rate of about 0.5°C./min and a very slow cooling rate of about 0.03° C./min. In anotherembodiment, narrow metastable cooling rates are observed at higherconcentrations (e.g., about 100 mg/mL).

The present disclosure provides morphic Form II of compound havingFormula II or III (or a pharmaceutically acceptable salt thereof) with aMSZW that results in lower solubility in methanol compared to polymorphsForm I and/or Form H. Lower solubility of Form II in methanol comparedto Form I of the present disclosure is consistent with Form II being themore stable form than Form I.

The crystallization of the present disclosure may include “seeding” inorder to achieve a desired final product size. Seeding also provides theability to obtain preferable polymorph form, obtain desired crystalmorphology, and obtain polymorphs or pseudo-polymorphs. In someembodiments of the present disclosure, crystallization process involvesseeding with Form I, II, or H of compound having Formula II or III (or apharmaceutically acceptable salt thereof). The present disclosureprovides Form II seeding during the crystallization process.

An embodiment of the present disclosure provides a morphic crystallineform (e.g., Form II) with an X-ray diffraction pattern includingprominent peaks at about 2.81 and about 5.63 degrees 2θ. In oneembodiment a composition comprising the morphic Form II of the compoundhaving Formula II or III is characterized by an X-ray diffractionpattern substantially similar to that set forth in FIG. 1 or FIG. 20,indexing substantially similar to that set forth in FIG. 2, and DSCThermogram substantially similar to that set forth in FIG. 4.

The present disclosure provides recrystallization of a crystalline form(e.g., Form II) of the compound having Formula II or III (Table 5). Inone embodiment of the present disclosure, primarily a single phase ofcrystalline Form II is provided, as evident from the XRPD pattern. Inone embodiment, ¹H NMR spectroscopy of the sample provides Formula II orIII as the chemical structure of the isolated crystalline Form II (FIG.6).

In certain embodiments, Form II exhibits an X-ray powder diffractionpattern having from two (2) to seven (7) characteristic peaks expressedin degrees 2θ at 2.81, 5.63, 19.00, 19.57, 22.76, and 24.70±0.2, orhaving an X-ray diffraction pattern substantially similar to that setforth in FIG. 1 or FIG. 20, indexing substantially similar to that setforth in FIG. 2, and DSC Thermogram substantially similar to that setforth in FIG. 4. In one embodiment, the XRPD peaks of Form II aresubstantially similar to that set forth in Table 6.

TABLE 6 Observed and Prominent Peaks for Form II Observed Peaks °2θ dspace (Å) Intensity (%)  2.81 ± 0.20 31.451 ± 2.410  100  5.63 ± 0.2015.688 ± 0.577  16 11.30 ± 0.20 7.832 ± 0.141 3 12.05 ± 0.20 7.345 ±0.124 2 13.22 ± 0.20 6.697 ± 0.102 2 13.45 ± 0.20 6.581 ± 0.099 5 13.81± 0.20 6.415 ± 0.094 4 14.32 ± 0.20 6.184 ± 0.087 5 14.92 ± 0.20 5.936 ±0.080 1 15.64 ± 0.20 5.665 ± 0.073 1 16.25 ± 0.20 5.456 ± 0.068 3 16.41± 0.20 5.401 ± 0.066 3 17.00 ± 0.20 5.217 ± 0.062 1 17.67 ± 0.20 5.021 ±0.057 6 17.87 ± 0.20 4.965 ± 0.056 5 18.15 ± 0.20 4.888 ± 0.054 6 18.35± 0.20 4.835 ± 0.053 4 18.50 ± 0.20 4.796 ± 0.052 3 19.00 ± 0.20 4.670 ±0.049 10 19.57 ± 0.20 4.536 ± 0.046 13 19.85 ± 0.20 4.472 ± 0.045 420.22 ± 0.20 4.391 ± 0.043 5 20.96 ± 0.20 4.239 ± 0.040 4 21.06 ± 0.204.219 ± 0.040 4 21.89 ± 0.20 4.060 ± 0.037 4 22.76 ± 0.20 3.907 ± 0.03410 23.70 ± 0.20 3.755 ± 0.032 8 23.95 ± 0.20 3.716 ± 0.031 4 24.32 ±0.20 3.660 ± 0.030 3 24.70 ± 0.20 3.604 ± 0.029 14 25.54 ± 0.20 3.488 ±0.027 3 26.12 ± 0.20 3.411 ± 0.026 1 26.52 ± 0.20 3.361 ± 0.025 1 26.81± 0.20 3.326 ± 0.025 1 27.07 ± 0.20 3.294 ± 0.024 1 27.48 ± 0.20 3.246 ±0.023 1 27.71 ± 0.20 3.220 ± 0.023 1 29.11 ± 0.20 3.067 ± 0.021 2 29.36± 0.20 3.042 ± 0.020 2 29.61 ± 0.20 3.017 ± 0.020 2 Prominent Peaks dspace (Å)  2.81 ± 0.20 31.451 ± 2.410  100  5.63 ± 0.20 15.688 ± 0.577 16

The skilled artisan recognizes that some variation is associated with2-theta (2θ) measurements. Typically, 2θ values may vary from ±0.1 to±0.2. The skilled artisan appreciates that such variation in values aregreatest with low 2θ values, and least with high 2θ values. The skilledartisan recognizes that different instruments may provide substantiallythe same XRPD pattern, even though the 2θ values vary somewhat.Moreover, the skilled artisan appreciates that the same instrument mayprovide substantially the same XRPD pattern for the same or differentsamples even though the XRPD of the respectively collected XRPD patternsvary slightly in the 2θ values. Such slight variation can be caused, forexample, by sample preparation techniques, different instruments used,instrument drift, and other experimental factors.

Diffraction peak lists may also be reported using d_(hkl) (observed peakpositions ° 20 may be converted into d_(hkl) values using Bragg's Law:d_(hkl)=λ/2 sin θ; Miller indices (hkl) of the diffraction peaks aredetermined from the published reference pattern, and when a referencepattern identifying (hkl) is unavailable, then “indexing” of the patternis needed to determine the (hkl)) and relative intensity rather than 20and absolute intensity. The peak position as 20 depends on instrumentalcharacteristics such as wavelength. The peak position as d_(hkl) is anintrinsic, instrument-independent material property. The absoluteintensity, i.e., the number of X-rays observed in a given peak, can varydue to instrumental and experimental parameters. To calculate relativeintensity, absolute intensity of every peak is divided by the absoluteintensity of the most intense peak, and then converted to a percentage.The most intense peak of a phase is therefore called the “100% peak.”Peak areas are a reliable measure of intensity.

The skilled artisan also appreciates that XRPD patterns of the samesample (taken on the same or different instruments) may exhibitvariations in peak intensity at the different 2θ values. The skilledartisan also appreciates that XRPD patterns of different samples of thesame polymorph (taken on the same or different instruments) may alsoexhibit variations in peak intensity at the different 2θ values. XRPDpatterns can be substantially the same pattern even though they havecorresponding 2θ signals that vary in their peak intensities.

In one embodiment, Form II exhibits an X-ray powder diffraction patternhaving two or more (e.g., three or more, four or more, five or more, sixor more, seven or more, eight or more, nine or more, or ten or more)characteristic peaks expressed in degrees 2θ (±0.2) at 2.81, 5.63,11.30, 12.05, 13.22, 13.45, 13.81, 14.32, 14.92, 15.64, 16.25, 16.41,17.00, 17.67, 17.87, 18.15, 18.35, 18.50, 19.00, 19.57, 19.85, 20.22,20.96, 21.06, 21.89, 22.76, 23.70, 23.95, 24.32, 24.70, 25.54, 26.12,26.52, 26.81, 27.07, 27.48, 27.71, 29.11, 29.36, and 29.61 or having anX-ray diffraction pattern substantially similar to that set forth inFIG. 1, FIG. 7, FIG. 13, FIG. 14, FIG. 20, FIG. 21, or FIG. 25, indexingsubstantially similar to that set forth in FIG. 2, and DSC Thermogramsubstantially similar to that set forth in FIG. 4.

In another embodiment, Form II exhibits an X-ray powder diffractionpattern having three or more characteristic peaks expressed in degrees2θ (±0.2) at 2.81, 5.63, 11.30, 12.05, 13.22, 13.45, 13.81, 14.32,14.92, 15.64, 16.25, 16.41, 17.00, 17.67, 17.87, 18.15, 18.35, 18.50,19.00, 19.57, 19.85, 20.22, 20.96, 21.06, 21.89, 22.76, 23.70, 23.95,24.32, 24.70, 25.54, 26.12, 26.52, 26.81, 27.07, 27.48, 27.71, 29.11,29.36, and 29.61 or having an X-ray diffraction pattern substantiallysimilar to that set forth in FIG. 1, FIG. 7, FIG. 13, FIG. 14, FIG. 20,FIG. 21, or FIG. 25, indexing substantially similar to that set forth inFIG. 2, and DSC Thermogram substantially similar to that set forth inFIG. 4.

In another embodiment, Form II exhibits an X-ray powder diffractionpattern having four or more characteristic peaks expressed in degrees 2θ(±0.2) at 2.81, 5.63, 11.30, 12.05, 13.22, 13.45, 13.81, 14.32, 14.92,15.64, 16.25, 16.41, 17.00, 17.67, 17.87, 18.15, 18.35, 18.50, 19.00,19.57, 19.85, 20.22, 20.96, 21.06, 21.89, 22.76, 23.70, 23.95, 24.32,24.70, 25.54, 26.12, 26.52, 26.81, 27.07, 27.48, 27.71, 29.11, 29.36,and 29.61 or having an X-ray diffraction pattern substantially similarto that set forth in FIG. 1, FIG. 7, FIG. 13, FIG. 14, FIG. 20, FIG. 21,or FIG. 25, indexing substantially similar to that set forth in FIG. 2,and DSC Thermogram substantially similar to that set forth in FIG. 4. Inanother embodiment, Form II exhibits an X-ray powder diffraction patternhaving characteristic peaks expressed in degrees 2θ (±0.2) at 2.81,5.63, 19.00, 19.57, 22.76, and 24.70, or having an X-ray diffractionpattern substantially similar to that set forth in FIG. 1, FIG. 7, FIG.13, FIG. 14, FIG. 20, FIG. 21, or FIG. 25, indexing substantiallysimilar to that set forth in FIG. 2, and DSC Thermogram substantiallysimilar to that set forth in FIG. 4, FIG. 15, FIG. 16, or FIGS. 22-24.

In a particular embodiment, Form II exhibits an X-ray powder diffractionpattern having at least eight characteristic peaks expressed in degrees2θ (±0.2), selected from the group consisting of at 2.81, 5.63, 11.30,12.05, 13.22, 13.45, 13.81, 14.32, 14.92, 15.64, 16.25, 16.41, 17.00,17.67, 17.87, 18.15, 18.35, 18.50, 19.00, 19.57, 19.85, 20.22, 20.96,21.06, 21.89, 22.76, 23.70, 23.95, 24.32, 24.70, 25.54, 26.12, 26.52,26.81, 27.07, 27.48, 27.71, 29.11, 29.36, and 29.61 or having an X-raydiffraction pattern substantially similar to that set forth in FIG. 1,FIG. 7, FIG. 13, FIG. 14, FIG. 20, FIG. 21, or FIG. 25, indexingsubstantially similar to that set forth in FIG. 2, and DSC Thermogramsubstantially similar to that set forth in FIG. 4, FIG. 15, or FIG. 16.

In another particular embodiment, Form II exhibits an X-ray powderdiffraction pattern having at least nine characteristic peaks expressedin degrees 2θ (±0.2), selected from the group consisting of at 2.81,5.63, 11.30, 12.05, 13.22, 13.45, 13.81, 14.32, 14.92, 15.64, 16.25,16.41, 17.00, 17.67, 17.87, 18.15, 18.35, 18.50, 19.00, 19.57, 19.85,20.22, 20.96, 21.06, 21.89, 22.76, 23.70, 23.95, 24.32, 24.70, 25.54,26.12, 26.52, 26.81, 27.07, 27.48, 27.71, 29.11, 29.36, and 29.61 orhaving an X-ray diffraction pattern substantially similar to that setforth in FIG. 1, FIG. 7, FIG. 13, FIG. 14, FIG. 20, FIG. 21, or FIG. 25,indexing substantially similar to that set forth in FIG. 2, and DSCThermogram substantially similar to that set forth in FIG. 4, FIG. 15,FIG. 16, or FIGS. 22-24.

In one or more embodiments, a composition comprising a compound of FormII lacks the XRPD peaks characteristic of Form H (e.g., at about 12.6°2θ) and/or Form I (e.g., at about 15.6° 2θ).

XRPD Indexing

XRPD patterns are indexed using X-Pert High Score Plus (v.2.2.1).Indexing and structure refinement computational studies were performed.Agreement between the allowed peak positions and the observed peaksindicates a consistent unit cell determination. Successful indexing of apattern indicates that the sample was composed primarily of a singlecrystalline phase. Space groups consistent with the assigned extinctionsymbol, unit cell parameters, and derived quantities are tabulated inthe respective figures providing the indexing solution for each form. Toconfirm the tentative indexing solution, the molecular packing motifswithin the crystallographic unit cells are determined.

XRPD Peak Identification

Under most circumstances, peaks within the range of up to about 30° 2θare selected. Rounding algorithms are used to round each peak to thenearest 0.1° or 0.01° 2θ, depending upon the instrument used to collectthe data and/or the inherent peak resolution. For d-space listings, thewavelength is used to calculate d-spacing was 1.541874 Å, a weightedaverage of the Cu-K_(α1) and Cu-K_(α2) wavelengths.

Variability associated with d-spacing estimates is calculated from theUSP recommendation, at each d-spacing, and provided in the respectivedata tables. Per USP guidelines, variable hydrates and solvates maydisplay peak variances greater than 0.2° 2θ and therefore peak variancesof 0.2° 2θ were not applicable to these materials. For samples with onlyone XRPD pattern and no other means to evaluate whether the sampleprovides a good approximation of the powder average, peak tables containdata identified only as “Prominent Peaks.” These peaks are a subset ofthe entire observed peak list. Prominent peaks were selected fromobserved peaks by identifying preferably non-overlapping, low-anglepeaks, with strong intensity.

Differential Scanning Calorimetry (DSC) Thermogram

Form II may also be identified based on DSC, indexing, SEM, and/orparticle size distribution. In some embodiments of the presentdisclosure, Form II is identifiable on the basis of a characteristicpeak observed in a differential scanning calorimetry thermogram.Differential scanning calorimetry, or DSC, is a thermoanalyticaltechnique in which the difference in the amount of heat required toincrease the temperature of a sample and reference is measured as afunction of temperature. In one embodiment, Form II exhibits adifferential scanning calorimetry thermogram showing a characteristicminor endotherm at about 43° C. (peak max) followed by overlapping majorendotherms at about 90 and about 95° C. (peak max). A final endothermwas observed with an onset at about 196° C. In another embodiment, FormII exhibits a differential scanning calorimetry thermogram substantiallyin accordance with FIG. 4.

In another embodiment of the invention, provided herein is Form IIcharacterized as a solid form of Compound 1, such that the solid formundergoes a weight increase of less than 1.5% upon increasing relativehumidity from 5.0% to 95.0%.

In one embodiment, Form II crystalline form of Formula II or III isprepared from a methanol crystallization using a slower cooling profileand no stirring. The DSC thermogram of Form II prepared using a slowercooling profile and no stirring has a minor endotherm at about 41° C.,overlapping endotherms at about 90 and about 95° C. (peak max), and afinal endotherm with an onset at about 200° C.

The present disclosure also provides a hydrate morphic Form H obtainedfrom a method including water during crystallization. The hydratemorphic Form H has a DSC characteristics of having a minor endothermaround 41° C. (peak max), major endotherm at about 70° C. and about 103°C. (peak max) and a final endotherm at about 193° C. The TGA of thehydrate morphic Form H is about 0.93% weight loss from about 25° C. toabout 100° C.

Scanning Electron Microscopy (SEM)

The present disclosure provides analysis of the crystals synthesized bythe disclosed method by SEM. Samples for SEM are prepared by placing asmall amount on a carbon adhesive tab supported on an aluminum mount.Each sample is then sputter coated with Au/Pd using a Cressington108auto Sputter Coater at approximately 20 mA and 0.13 mbar (Ar). Eachsample is observed under high vacuum using a beam voltage of 5.0 kV.

In some embodiments, SEM is performed using a FEI Quanta 200 scanningelectron microscope equipped with an Everhart Thornley (ET) detector.Images are collected and analyzed using xTm (v. 2.01) and XT Docu (v.3.2) software, respectively. The magnification is verified using aNIST-traceable standard. The sample is prepared for analysis by placinga small amount on a carbon adhesive tab supported on an aluminum mount.The sample is then sputter coated twice with Au/Pd using a Cressington108auto Sputter Coater at approximately 20 mA and 0.13 mbar (Ar) for 75seconds (FIGS. 26-32).

TABLE 7 Characteristics of morphic Form II XRPD Crystalline; designatedForm II Indexing Primitive Monoclinic a = 7.537 Å, b = 6.729 Å, c =62.555 Å, α = 90°, β = 90.54°, γ DSC Minor endo max. at ~43° C.;overlapping endo at ~90° C. and ~95° C. (max). Endo onset at SEM Largeagglomerates (Agglomerates of plates and tablets (FIGS. 26-29)) PSA d10= 19.896 μm; d50 = 101.745 μm; d90 = (Sample 3) 274.113 μm; bimodaldistribution ¹H NMR Consistent with Compound 1 structure; possiblycontains residual acetone (wash solvent)

Purity

In certain embodiments, a sample of Form II contains impurities.Non-limiting examples of impurities include amorphous forms, otherpolymorph forms (e.g., H, I, and III), or residual organic and inorganicmolecules such as related impurities (e.g., intermediates used to makeForm II or fragments thereof), solvents, water or salts. In oneembodiment, a sample of Form II is substantially free from impurities,meaning that no significant amount of impurities is present. In anotherembodiment, a sample of Form II contains less than 10% by weight totalimpurities. In another embodiment, a sample of Form II contains lessthan 5% by weight total impurities. In another embodiment, a sample ofForm II contains less than 1% by weight total impurities. In yet anotherembodiment, a sample of Form II contains less than 0.1% by weight totalimpurities.

The present disclosure provides that an anhydrous form (e.g. Form II) isproduced without forming a hydrate form (e.g., Form H) during thecrystallization process, by determining the critical water concentrationbelow which the anhydrous form is the stable form. The critical waterconcentration is determined by varying the methanol-water concentrationsin the slurries.

In one embodiment, in sub-ambient slurries, at 99:1 methanol:water, FormII was recovered. In another embodiment, at concentrations of 97:3methanol:water and greater, Form H was recovered from the slurries.These embodiments provide that the critical water concentration forforming Form II is between 1 and 3% water. In some embodiments, water inthe initial methanol solvent is not accounted for, which results inslightly higher final water concentration than 1 and 3%. In additionalembodiments, experiments, by varying the methanol-water concentrationsin the slurries, is carried out at room temperature or at about 45° C.In one embodiment, the critical water concentration is less than 5% atroom temperature and less than 10% at about 45° C. Mixtures of Form IIand Form H are observed to convert to either Form II or Form H inmethanol depending on the exact water content. The present disclosureprovides that during crystallization of Form II, critical water activityremains very low up to about 45° C. and that water content in the finalmethanol recrystallization is a critical process parameter to control toavoid operating in conditions which favors formation of Form H.

In certain embodiments, a sample of Form II is a crystalline solidsubstantially free of amorphous compound of Formula II or III. As usedherein, the term “substantially free of amorphous compound of Formula IIor III” means that the compound contains no significant amount ofamorphous compound of Formula II or III. In another embodiment, a sampleof crystalline compound of Formula II or III comprises Form IIsubstantially free of Form I and/or H. As used herein, the term“substantially free of Form I and/or H” means that a sample ofcrystalline compound of Formula II or III contains no significant amountof Form I and/or H. In certain embodiments, at least about 90% by weightof a sample is Form II, with only 10% being Form I and/or H and/oramorphous compound of Formula II or III. In certain embodiments, atleast about 95% by weight of a sample is Form II, with only 5% beingForm I and/or H and/or amorphous compound of Formula II or III. In stillother embodiments of the invention, at least about 99% by weight of asample is Form II, with only 1% by weight being Form I and/or H and/oramorphous compound of Formula II or III. In still other embodiments ofthe invention, at least about 99.5% by weight of a sample is Form II,with only 0.5% by weight being Form I and/or H and/or amorphous compoundof Formula II or III. In still other embodiments of the invention, atleast about 99.9% by weight of a sample is Form II, with only 0.1% byweight being Form I and/or H and/or amorphous compound of Formula II orIII.

Form II may occur as any reasonable tautomer, or a mixture of reasonabletautomers. As used herein, “tautomer” refers to one of two or morestructural isomers that exist in equilibrium and are readily convertedfrom one isomeric form to another.

In some embodiments, the process of recrystallizing Compound 1 (e.g.,Formula II or Formula III) results in a Compound 1 that is largely freeof impurities. In some embodiments, Compound 1 is >95% pure, >96%pure, >97% pure, >98% pure, >99% pure, >99.5% pure, >99.9% pure,or >99.99% pure. The purity can be measured by a variety of differenttechniques known in the art such as high-performance liquidchromatography (HPLC). For instance, in some preferred embodiments,Compound 1 is recrystallized once from methanol and heptane, followed bythree subsequent recrystallizations from methanol; Form II is onlyseeded during the third and final methanol recrystallization.

The recrystallization protocol for Compound 1 (e.g., Compound 1 Form II)can impact the relative purity of the Compound 1. For instance, FIG. 27shows a CAD (Charged Aerosol Detection) chromatogram of Compound 1 aftertwo fast-cool-down methanol recrystallizations. For instance, theCompound 1 is 98.5% pure. As indicated in FIG. 27.

FIG. 28 shows a CAD chromatogram of Compound 1 after three slowcool-down recrystallizations in methanol. As shown in FIG. 28, theCompound 1 is 99.5% pure.

FIG. 29 shows a CAD chromatogram of Compound 1 after onerecrystallization in heptane/methanol followed by one slow cool-downrecrystallization in methanol. The compound is 100% pure as shown inFIG. 29.

FIG. 30 shows a CAD chromatogram of Compound 1 after a preferredembodiment comprising one recrystallization from heptane/methanolfollowed by three recrystallizations from methanol. As shown in FIG. 30,the purity of Compound 1 is 100%. This recrystallization process isexplained in more detail in Example 3 and is a preferred purificationprotocol.

Accordingly, in some preferred embodiments, the process of purifying theCompound 1 comprises five distinct recrystallization procedures: threemethanol recrystallizations without seeding, one n-heptane/methanolrecrystallization, and one methanol recrystallization with seeding. Insome embodiments, the first three methanol recrystallizations removeimpurities such as residual amounts of the following compounds (i.e.,Compounds A and B):

In some embodiments, the above compounds can be substantially difficultto remove from the compositions comprising Compound 1 by other means(e.g., chromatography). Accordingly, in some embodiments, it isadvantageous to perform at least one recrystallization from methanol asa first recrystallization procedure in order to remove the aboveimpurities.

The recrystallization using n-heptane and methanol removes theimpurities detectable by CAD and shown in FIGS. 27 and 28. In someembodiments, the CAD-detectable impurities can be, for instance, thefollowing compounds (i.e., Compounds C and D):

Accordingly, in some embodiments in the absence of performing the aboverecrystallizations, the compounds shown above will be present in thecomposition comprising Compound 1. The above compounds in anycombination can be present in low quantities (e.g., <2% by weight, or<1% by weight).

In some embodiments, the present invention includes a compositioncomprising Form II and various other morphic forms. The other morphicforms can include Form I and Form H. In some embodiments, thecomposition includes >90% Form II. In some embodiments, the compositionincludes >95% Form II. In some embodiments, the compositionincludes >99% Form II. Alternatively, in some embodiments, thecomposition includes <10% of Form I and/or Form H. In some embodiments,the composition includes <5% of Form I and/or Form H. In someembodiments, the composition includes <1% of Form I and/or Form H.

In some embodiments, the present invention includes a compositioncomprising Compound 1 and other impurities. In some embodiments, theimpurities are selected from Compounds A-D. In some embodiments, thepresent invention includes a composition comprising >90% Compound 1. Insome embodiments, the present invention includes a compositioncomprising >95% Compound 1. In some embodiments, the present inventionincludes a composition comprising >99% Compound 1. In some embodiments,the present invention includes a composition comprising <10% impuritiesselected from Compounds A-D. In some embodiments, the present inventionincludes a composition comprising <5% impurities selected from CompoundsA-D. In some embodiments, the present invention includes a compositioncomprising <1% impurities selected from Compounds A-D.

Co-Crystals

The present disclosure provides co-crystals of Form II of Formula II orIII. The co-crystals of the present disclosure may be a pharmaceuticallyacceptable salt of Formula II or III, a solvate (a crystal structureincorporating either stoichiometric or non-stoichiometric amounts of asolvent), hydrate (a crystal structure incorporating eitherstoichiometric or non-stoichiometric amounts of water), clathrate(molecules of one substance are completely enclosed with the crystalstructure of another), and/or molecular complex (a unique crystalstructure incorporating stoichiometric amounts of more than onemolecule).

Co-crystal may be formed with citric acid, fumaric acid, gentisic acid,hippuric acid, maleic acid, L-mandelic acid, orotic acid, oxalic acid,saccharin, succinic acid, L-tartaric acid, toluenesulfonic acid,ammonia, L-arginine, calcium hydroxide, diethylamine,diethylaminoethanol, ethylenediamine, 1H imidazole, L-lysine,2-hydroxyethylmorpholine, N-methyl-glucamine, potassium methanolate,zinc tert-butoxide. The salt/co-crystal screening included theevaporation from four solvents followed by the phase equilibration infour further solvents.

Method of Synthesis Morphic Form II

The present invention provides methods for the synthesis of thecompounds of Formulae I, II, and/or II. The present invention alsoprovides detailed methods for the synthesis of various disclosedcompounds of the present invention according to the following schemesand as shown in the Examples.

Throughout the description, where compositions are described as having,including, or comprising specific components, it is contemplated thatcompositions also consist essentially of, or consist of, the recitedcomponents. Similarly, where methods or processes are described ashaving, including, or comprising specific process steps, the processesalso consist essentially of, or consist of, the recited processingsteps. Further, it should be understood that the order of steps or orderfor performing certain actions is immaterial so long as the inventionremains operable. Moreover, two or more steps or actions can beconducted simultaneously.

The synthetic processes of the invention can tolerate a wide variety offunctional groups; therefore various substituted starting materials canbe used. The processes generally provide the desired final compound ator near the end of the overall process, although it may be desirable incertain instances to further convert the compound to a pharmaceuticallyacceptable salt, ester or derivative thereof.

Scheme 1 (Steps 1, 2A and 2B): Synthesis of phosphonic acid,[[(S)-2-(4-amino-2-oxo-1(2H)-pyrimidinyl)-1-(hydroxymethyl)ethoxy]methyl]mono[3-(hexadecyloxy)propyl]ester (Compound 1).

In one embodiment, the present disclosure provides a method of producingmorphic Form II of the compound of formula II or formula III, or apharmaceutically acceptable salt thereof. The method provides apurification process including recrystallizing a preparation ofphosphonic acid,[[(S)-2-(4-amino-2-oxo-1(2H)-pyrimidinyl)-1-(hydroxymethyl)ethoxy]methyl]mono[3-(hexadecyloxy)propyl]ester (Compound 1) frommethanol.

The present disclosure provides a method for synthesizing a morphic FormII of Compound 1, including the steps 1 and 2.

Step 1:

Synthesis of (S)—N¹-[(2-hydroxy-3-triphenylmethoxy)propyl]cytosine(Compound 2).

(S)—N¹-[(2-hydroxy-3-triphenylmethoxy)propyl]cytosine (Compound 2) issynthesized by contacting cytosine with (S)-trityl glycidyl ether (syn.(S)-GLYCIDYL TRITYL ETHER; Trityl-(s)-glycidyl ether; (S)-TRITYLGLYCIDYL ETHER; (S)-(−)-TRITYL GLYCIDYL ETHER; (S)-(−)-GLYCIDYL TRITYLETHER; Triphenylmethyl glycidyl ether; (S)-2-((trityloxy)methyl)oxirane;(S)-(−)-Glycidyl Trityl Esther; (S)-GLYCIDYL TRIPHENYLMETHYL ETHER;(S)-2-(TRIPHENYLMETHOXYMETHYL)OXIRANE) in the presence of a suitablebase such as a metal carbonate (e.g., potassium carbonate) in a suitableorganic solvent (e.g., N,N-dimethylformamide (DMF) or tert-amyl alcohol)in a suitable reaction temperature (e.g., about 85 to about 95° C. orabout 60 to about 120° C.) until completion of reaction for betweenabout 4 to 14 hours, e.g., about 8 to 10 hours. In one embodiment, thereaction can be heated at a temperature between about 85 to about 95° C.for about 9 hours.

The heated reaction mixture is then cooled. In one embodiment, theheated reaction mixture is cooled to, e.g., about 50-75° C. or about66-70° C., and quenched with a substituted benzene derivative, e.g., amono-substituted benzene derivative such as toluene. The resultingslurry is further cooled to a temperature, e.g., between below or closeto 0° C., e.g., to about −10 to 5° C. The cooled slurry is thenfiltered, washed with a substituted benzene derivative, e.g., amono-substituted benzene derivative such as toluene. The solids obtainedafter washing in a substituted benzene derivative, e.g., amono-substituted benzene derivative such as toluene, is then made into aslurry at a suitable temperature, e.g., about 15-25° C., before theslurry is filtered. The cooled slurry is then washed with an organicsolvent such as a ketone, e.g., acetone (propanone).

The solid is then purified by trituration in water/acetone at a suitableratio (e.g., 90.0 kg/54.0 kg) at a suitable temperature, e.g., about17-22° C., filtered, and washed with an organic solvent such as aketone, e.g., acetone (propanone) (e.g., about 36.0 kg). The embodimentsprovide that the filter cake obtained after the filtration step is thensuspended in an organic solvent such as a ketone, e.g., acetoneropanone) (e.g., about 178.9 kg) and heated, e.g., at approximately35-45° C. for more than 1 hour, e.g., equal to or more than about 1.1,1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5,2.6, 2.7, 2.8, 2.9 or 3.0 hours, and then filtered, and washed with anorganic solvent such as a ketone, e.g., acetone (propanone) (e.g., about36.0 kg). The washes and triturations are repeated as needed to removeresidues and/or impurities, e.g., residual cytosine and/orprocess-related impurities. The cake is dried in vacuo at equal to orless than about 40° C. for several hours, e.g., 12 hours, to yield about45.0 kg (about 65.0%) of Compound 2. In some embodiments, purity of theyield is more than about 99% (as determined by HPLC (AUC)). In oneembodiment, ¹H-NMR of the product is consistent with the standardstructure of Compound 2.

Steps 2A and 2B:

Synthesis of phosphonic acid,[[(S)-2-(4-amino-2-oxo-1(2H)-pyrimidinyl)-1-(hydroxymethyl)ethoxy]methyl]mono[3-(hexadecyloxy)propyl]ester (Compound 1), having theformula:

The Formula II or III compound is prepared by contacting Compound 2 withCompound 4 in the presence of a suitable base such as a metal alkoxide(e.g., magnesium di-tert-butoxide, sodium tert-butoxide, lithiumtert-butoxide, sodium tert-amyl alkoxide, potassium tert-butoxide,sodium methoxide), metal hydride (e.g., sodium hydride, potassiumhydride), or metal amide (e.g., lithium bis(trimethylsilyl)amide) in asuitable organic solvent (e.g., N,N-dimethylformamide,N,N-dimethylacetamide, dimethylsulfoxide, 1-methyl-2-pyrrolidinone) at asuitable reaction temperature (e.g., 50 to 110° C.) until completion ofreaction, which is about 0.25 to five hours, e.g., about two to fourhours. The crude reaction mixture is subjected to an aqueous work-up.The crude product is extracted with a suitable organic solvent (e.g.,ethyl acetate, isopropyl acetate, dichloromethane, etc.) and the organicsolvent is concentrated to give crude Compound 3. The concentratecontaining Compound 3 is diluted in methanol and reconcentrated toremove residual organic solvent (e.g., ethyl acetate, isopropyl acetate,dichloromethane, etc.). The crude Compound 3 is contacted with asuitable deprotecting agent (e.g., hydrogen chloride, acetyl chloride)in an organic solvent (e.g., methanol) until completion of reaction, forone or more hours, e.g., one to six hours. In one embodiment, Compound 3is contacted with a suitable deprotecting agent (e.g., hydrogenchloride, acetyl chloride) in an organic solvent (e.g., methanol) untilcompletion of reaction for two to three hours. The crude Compound 1 isrecrystallized using a suitable solvent system (e.g.,methanol/acetone/water, ethanol, methanol).

In one embodiment a mixture of(S)—N¹-[(2-hydroxy-3-triphenylmethoxy)propyl]cytosine (Compound 2),P-[[[4-methylphenyl)sulfonyl]oxy]methyl]-,mono[3-(hexadecyloxy)propyl]ester, sodium salt (Compound 4), magnesiumtert-butoxide, and a polar (hydrophilic) aprotic solvent, e.g.,dimethylformamide (DMF), is heated at a suitable temperature, e.g.,between about 75-85° C. for more than 1 hour, e.g., equal to or morethan about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2,2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9 or 3.0 hours. Other polar aproticsolvent such as tetrahydrofuran, ethyl acetate, acetone, acetonitrile,or dimethyl sulfoxide is also used in the mixture.

The mixture solution is then cooled to a suitable temperature, e.g.,about 25-35° C., before adding an organic solvent, e.g., an ester suchas isopropyl acetate (1-Methylethyl acetate). The solution is thenfurther cooled to a suitable temperature, e.g., about 15-25° C. beforewashing sequentially with an acid solution, e.g., HCl solution, and asalt solution, e.g., NaCl solution.

The organic solvent e.g., an ester such as isopropyl acetate(1-Methylethyl acetate) is removed by vacuum distilling the organicphase to form a concentrate. The concentrate is then diluted with analcohol (e.g., methanol) to further remove isopropyl acetate and tore-concentrate and form a mixture containing phosphonic acid,[[(S)-2-(4-amino-2-oxo-1(2H)-pyrimidinyl)-1-(hydroxymethyl)-2-(triphenylmethoxy)ethyl]methyl]mono[3-(hexadecyloxy)propyl]ester(Compound 3). The temperature is maintained between about −5 and 15° C.by the addition of HCl gas at a desired rate. The reaction is maintainedbelow about 10-20° C. for more than 1 hour, e.g., about 2 hours, beforeit is filtered to remove solid impurities. The filtrate is then dilutedwith water and pH is then adjusted to about 2.3-2.7 with NaOH. Thesolids are filtered and washed with water before a slurry is prepared inan organic solvent such as a ketone, e.g., acetone (propanone) at about35-45° C. for about 1 hour. The slurry is then filtered and washed withan organic solvent such as a ketone, e.g., acetone (propanone). Theorganic solvent such as a ketone, e.g., acetone (propanone) washed crudeproduct is then dried at equal to or less than about 40° C. for severalhours, e.g., about 12 hours and heated at about 60-70° C. in alcohol(e.g., methanol). The dried crude product is then polish filtered,cooled to about 58-62° C., stirred for one hour, cooled to first about48-52° C. for about six hours, then to about 17-23° C. for two hours,filtered, and then washed with alcohol (e.g., methanol). The washing,drying, polish filtering, cooling, and the final filtering and washingsteps may be repeated one or more times to dissolve solids in alcohol(e.g., methanol) before the sample is cooled to, e.g., about 59-61° C.The cooled product is then stirred for several minutes, e.g., about 20minutes. To this solution a seed stock of phosphonic acid,[[(S)-2-(4-amino-2-oxo-1(2H)-pyrimidinyl)-1-(hydroxymethyl)ethoxy]methyl]mono[3-(hexadecyloxy)propyl]ester (e.g. seed stock ofabout 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%,about 0.7%, about 0.8%, about 0.9%, about 1.0%, about 1.1%, about 1.2%,about 1.3%, about 1.4%, about 1.5%, about 1.6%, about 1.7%, about 1.8%,about 1.9%, about 2.0%, about 2.1%, about 2.2%, about 2.3%, about 2.4%,about 2.5%, about 2.6%, about 2.7%, about 2.8%, about 2.9%, about 3.0%,about 3.1%, about 3.2%, about 3.3%, about 3.4%, about 3.5%, about 3.6%,about 3.7%, about 3.8%, about 3.9%, about 4.0%, about 4.1%, about 4.2%,about 4.3%, about 4.4%, about 4.5%, about 4.6%, about 4.7%, about 4.8%,about 4.9%, about 5.0%, about 5.1%, about 5.2%, about 5.3%, about 5.4%,about 5.5%, about 5.6%, about 5.7%, about 5.8%, about 5.9%, about 6.0%,about 6.1%, about 6.2%, about 6.3%, about 6.4%, about 6.5%, about 6.6%,about 6.7%, about 6.8%, about 6.9%, about 7.0%, about 8.0%, about 9.0%,about 10.0%, about 0.1-0.2%, about 0.2-0.3%, about 0.3-0.4%, about0.4-0.5%, about 0.5-0.6%, about 0.6-0.7%, about 0.7-0.8%, about0.8-0.9%, about 0.9-1.0%, about 1.0-2.0%, about 2.0-3.0%, about3.0-4.0%, about 4.0-5.0%, about 5.0-6.0%, about 6.0-7.0%, about7.0-8.0%, about 8.0-9.0%, about 9.0-10.0%, about 7.0-8.0%, about8.0-9.0%, or about 9.0-10.0% of Form I, Form II, or Form H) is added andthen stirred for more than 1 hour, e.g., about 2 hours, and then thesolution is cooled to about 47° C.-53° C. by stirring for about severalhours, e.g., 8 hours, and then stirred for about more than one hour,e.g., about 2 hours. The seed is added at a temperature between about50° C.-65° C. (e.g., 56° C.-61° C.). The stirred solution is thenfurther cooled to about 17-23° C. over at least about 1 hour, e.g.,about 6 hours or more, and further stirred for about 2 hours; filteredand washed with methanol; dried at about equal to or less than 40° C.for about 24 hours. The method of the present disclosure therebyprovides synthesis of morphic Form II of phosphonic acid,[[(S)-2-(4-amino-2-oxo-1(2H)-pyrimidinyl)-1-(hydroxymethyl)ethoxy]methyl]mono[3-(hexadecyloxy)propyl]ester (Compound 1).

In some embodiments, a composition of morphic Form II can include thecorresponding hydrate (i.e., Form H). In some embodiments, a compositionof Form II has trace amounts (e.g., <1.5%, <1%, <0.5%, <0.4%, <0.3%,<0.2%, <0.1%, or 0.01% of Form H (e.g., the composition is substantiallyfree of Form H). For instance, the XRPD pattern of Compound 1 shown inFIG. 20 exhibits sharp peaks indicating the sample is composed ofcrystalline material. The pattern is similar to sample 1 of Form II interms of peak positions (see e.g., FIG. 1), suggesting the sample iscomposed of Form II. However, the peak at ˜12.6° 2θ may be due to thepresence of a small amount of Compound 1, Form H (see e.g., FIG. 3).

In some embodiments (for instance, sample 3), The DSC thermogram of FormII can display a small endotherm with a maximum at ˜42° C. (onset ˜42°C.), overlapping endotherms with maxima at ˜90° C. and ˜97 (onsets ˜89and 96° C., respectively), and an endotherm at ˜202° C. (onset ˜201° C.)(see e.g., FIG. 22-24). The thermogram is similar to sample 1 ofCompound 1, Form II (FIG. 4). However, a minor endotherm is observed at105° C. in the scan of sample 3, Form II (FIGS. 22-24). This may be dueto the presence of a minor amount of Form H.

For instance, sample 3 of Compound 1, Form II has the following DSCprofile: small, sharp endotherm: maximum ˜42° C. (onset ˜42° C.);Overlapping sharp endotherms: maxima ˜90° C. (onset ˜89° C.); maxima˜97° C. (onset ˜96° C.); Minor endotherm: ˜105° C.; Sharp endotherm:maximum ˜202° C. (onset ˜201° C.). This profile is consistent with FormII plus an additional trace amount of a minor endotherm ˜105° C. whichcan be due to the presence of a trace amount of Form H.

The present disclosure provides more than about 99% wt/wt pure morphicForm II of a compound having Formula II or III, or a pharmaceuticallyacceptable salt thereof. In some embodiments, a composition comprisingthe morphic Form II of a compound having Formula II or III, or apharmaceutically acceptable salt thereof is equal to or more than about99% wt/wt, about 98% wt/wt, about 97% wt/wt, about 96% wt/wt, about 95%wt/wt, about 94% wt/wt, about 93% wt/wt, about 92% wt/wt or about 91%wt/wt, pure. In some embodiments, the present disclosure provides anon-hydrate morphic Form II of a compound having Formula II or III, or apharmaceutically acceptable salt thereof. In some embodiments, acomposition comprising the morphic Form II of the present disclosure isequal to or less than about 5 mg/mL, about 4 mg/mL, about 3 mg/mL, about2 mg/mL, or about 1 mg/mL soluble in 1:1 methanol:water ratio at roomtemperature and less than about 20 mg/mL, about 19 mg/mL, about 18mg/mL, about 17 mg/mL, about 16 mg/mL, about 15 mg/mL, about 14 mg/mL,about 13 mg/mL, about 12 mg/mL, about 11 mg/mL, or about 10 mg/mLsoluble at a temperature higher than the room temperature, e.g., atabout 63° C. In some embodiments, a composition comprising the morphicForm II of the present disclosure is characterized by an X-raydiffraction pattern including prominent peaks at about 2.81 and about5.63 degrees 2θ. In one embodiment, a composition comprising the morphicForm II synthesized and/or crystallized by the disclosed method ischaracterized by an X-ray diffraction pattern substantially similar tothat set forth in FIG. 1. In yet another embodiment, a compositioncomprising the morphic Form II synthesized and/or crystallized by thedisclosed method is characterized by an X-ray diffraction patternsubstantially similar to that set forth in FIG. 13 or 17. The presentdisclosure also provide that morphic Form II synthesized and/orcrystallized by the disclosed method shows a minor endotherm at about41-43° C. (peak max) followed by overlapping major endotherms at about90 and about 95° C. (peak max) in a DSC thermogram. In one embodiment,the final endotherm of morphic Form II synthesized and/or crystallizedby the disclosed method has an onset at about 196° C. in a DSCthermogram.

The present disclosure provides a method for synthesizing crystallinemorphic Form II of phosphonic acid,[[(S)-2-(4-amino-2-oxo-1(2H)-pyrimidinyl)-1-(hydroxymethyl)ethoxy]methyl]mono[3-(hexadecyloxy)propyl]ester, by seeding with about0.1-10% (e.g., about 0.1-0.2%, about 0.2-0.3%, about 0.3-0.4%, about0.4-0.5%, about 0.5-0.6%, about 0.6-0.7%, about 0.7-0.8%, about0.8-0.9%, about 0.9-1.0%, about 1.0-2.0%, about 2.0-3.0%, about3.0-4.0%, about 4.0-5.0%, about 5.0-6.0%, about 6.0-7.0%, about7.0-8.0%, about 8.0-9.0%, or about 9.0-10.0%) of seed of the phosphonicacid. Some embodiments provide seeding the crystallization process withabout 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%,about 0.7%, about 0.8%, about 0.9%, about 1.0%, about 1.1%, about 1.2%,about 1.3%, about 1.4%, about 1.5%, about 1.6%, about 1.7%, about 1.8%,about 1.9%, about 2.0%, about 2.1%, about 2.2%, about 2.3%, about 2.4%,about 2.5% about 2.6%, about 2.7%, about 2.8%, about 2.9%, about 3.0%,about 3.1%, about 3.2%, about 3.3%, about 3.4%, about 3.5%, about 3.6%,about 3.7%, about 3.8%, about 3.9%, about 4.0%, about 4.1%, about 4.2%,about 4.3%, about 4.4%, about 4.5%, about 4.6%, about 4.7%, about 4.8%,about 4.9%, about 5.0%, about 5.1%, about 5.2%, about 5.3%, about 5.4%,about 5.5%, about 5.6%, about 5.7%, about 5.8%, about 5.9%, about 6.0%,about 6.1%, about 6.2%, about 6.3%, about 6.4%, about 6.5%, about 6.6%,about 6.7%, about 6.8%, about 6.9%, about 7.0%, about 7.0-about 8.0%,about 8.0-about 9.0%, or about 9.0%-about 10% of seed of the phosphonicacid. In additional embodiments the crystalline morphic form is formedwith slow or medium stirring or agitation during the crystallizationprocess. In one embodiment, the rate of stirring affects the type ofmorphic form produced. In another embodiment, the rate of stirring doesnot affect the type of morphic form produced. The embodiments providemorphic Form II crystallization by a process comprising methanol. Theembodiments further provide crystallization process for forming morphicForm II by seeding with Form II or Form I of the phosphonic acid.

In some embodiments, Form II of Compound 1 is substantially free ofimpurities. The present disclosure provides, a morphic Form II of acompound having Formula II or III having a purity of equal to or greaterthan 91% is used to treat viral infection (e.g., a dsDNA viralinfection) in a subject wherein said infection is resistant tovalganciclovir hydrochloride (or ganciclovir) or wherein said subjectexhibits side effects to valganciclovir hydrochloride (or ganciclovir).Alternatively or additionally, the compound of Formula II or III havinga purity of equal to or greater than 91% wt/wt or being in Form II,e.g., having less than or equal to 9% wt/wt of other morphic forms or anamorphous form, is used to treat cytomegalovirus (CMV) subsequent totreatment with ganciclovir, for example, wherein the CMV infection isemergent. The patient may be a bone marrow stem cell transplant patient,especially where there is a risk (real or perceived) for bone marrowtoxicity from ganciclovir in the patient.

In another embodiment, the subject is a mammal. In another embodiment,the subject is a human.

In another embodiment, a compound of Formula II or III having a purityof equal to or greater than 91% or being in Form II is administeredorally, for example, at a dosage of about 0.01 mg/kg to about 10 mg/kgor more, e.g., up to about 100 mg/kg. In another embodiment, saidcompound of Formula II or III having a purity of equal to or greaterthan 91% wt/wt or being in Form II, e.g., having less than or equal to9% wt/wt of other morphic forms or an amorphous form, is administered tosaid subject at a dosage of about 0.01 mg/kg, about 0.05 mg/kg, about0.1 mg/kg, about 0.5 mg/kg, about 1.0 mg/kg, about 1.5 mg/kg, about 2.0mg/kg, about 2.5 mg/kg, about 3.0 mg/kg, about 3.5 mg/kg, about 4.0mg/kg, about 4.5 mg/kg, about 5.0 mg/kg, about 5.5 mg/kg, about 6.0mg/kg, about 6.5 mg/kg, about 7.0 mg/kg, about 7.5 mg/kg, about 8.0mg/kg, about 8.5 mg/kg, about 9.0 mg/kg, about 9.5 mg/kg, or about 10mg/kg or more or any range therein.

The present disclosure provides, morphic Form II of a compound ofFormula II or III (or a pharmaceutically acceptable salt thereof)characterized by an X-ray diffraction pattern with two or more (e.g.,three or more, four or more, five or more, six or more, seven or more,eight or more, nine or more, or ten or more) peaks expressed in degrees2θ (±0.2) selected from 2.81, 5.63, 11.30, 12.05, 13.22, 13.45, 13.81,14.32, 14.92, 15.64, 16.25, 16.41, 17.00, 17.67, 17.87, 18.15, 18.35,18.50, 19.00, 19.57, 19.85, 20.22, 20.96, 21.06, 21.89, 22.76, 23.70,23.95, 24.32, 24.70, 25.54, 26.12, 26.52, 26.81, 27.07, 27.48, 27.71,29.11, 29.36, and 29.61, or having an X-ray diffraction patternsubstantially similar to that set forth in FIG. 1, FIG. 7, FIG. 13, FIG.14, FIG. 20, FIG. 21, or FIG. 25, administered at a dose of about 100 mgtwice a week or at a dose of about 200 mg once a week. In oneembodiment, a composition comprising the morphic Form II having indexingsubstantially similar to that set forth in FIG. 2 and/or a DSCThermogram substantially similar to that set forth in FIG. 4, FIG. 15,FIG. 16 or FIGS. 22-24 is administered at a dose of about 100 mg twice aweek or at a dose of about 200 mg once a week.

The present disclosure provides, morphic Form II of a compound ofFormula II or III (or a pharmaceutically acceptable salt thereof)characterized by an X-ray diffraction pattern with two or more (e.g.,three or more, four or more, five or more, six or more, seven or more,eight or more, nine or more, or ten or more) peaks expressed in degrees2θ (±0.2) selected from 2.81, 5.63, 11.30, 12.05, 13.22, 13.45, 13.81,14.32, 14.92, 15.64, 16.25, 16.41, 17.00, 17.67, 17.87, 18.15, 18.35,18.50, 19.00, 19.57, 19.85, 20.22, 20.96, 21.06, 21.89, 22.76, 23.70,23.95, 24.32, 24.70, 25.54, 26.12, 26.52, 26.81, 27.07, 27.48, 27.71,29.11, 29.36, and 29.61, or having an X-ray diffraction patternsubstantially similar to that set forth in FIG. 1, FIG. 7, FIG. 13, FIG.14, FIG. 20, FIG. 21, or FIG. 25, administered at a dose of about 1-4mg/kg (e.g., about 1-1.1 mg/kg, about 1.1-1.2 mg/kg, about 1.2-1.3mg/kg, about 1.3-1.4 mg/kg, about 1.4-1.5 mg/kg, about 1.5-1.6 mg/kg,about 1.6-1.7 mg/kg, about 1.7-1.8 mg/kg, about 1.8-1.9 mg/kg, about1.9-2.0 mg/kg, about 2.0-2.1 mg/kg, about 2.1-2.2 mg/kg, about 2.2-2.3mg/kg, about 2.3-2.4 mg/kg, about 2.4-2.5 mg/kg, about 2.5-2.6 mg/kg,about 2.6-2.7 mg/kg, about 2.7-2.8 mg/kg, about 2.8-2.9 mg/kg, about2.9-3.0 mg/kg, about 3.0-3.1 mg/kg, about 3.1-3.2 mg/kg, about 3.2-3.3mg/kg, about 3.3-3.4 mg/kg, about 3.4-3.5 mg/kg, about 3.5-3.6 mg/kg,about 3.6-3.7 mg/kg, about 3.7-3.8 mg/kg, about 3.8-3.9 mg/kg, or about3.9-4.0 mg/kg).

In some embodiments, a composition comprising the morphic Form II havingindexing substantially similar to that set forth in FIG. 2 and/or a DSCThermogram substantially similar to that set forth in FIG. 4, FIG. 15,FIG. 16 or FIGS. 22-24 is administered at a dose of about 1-4 mg/kg(e.g., about 1.0-1.1 mg/kg, about 1.1-1.2 mg/kg, about 1.2-1.3 mg/kg,about 1.3-1.4 mg/kg, about 1.4-1.5 mg/kg, about 1.5-1.6 mg/kg, about1.6-1.7 mg/kg, about 1.7-1.8 mg/kg, about 1.8-1.9 mg/kg, about 1.9-2.0mg/kg, about 2.0-2.1 mg/kg, about 2.1-2.2 mg/kg, about 2.2-2.3 mg/kg,about 2.3-2.4 mg/kg, about 2.4-2.5 mg/kg, about 2.5-2.6 mg/kg, about2.6-2.7 mg/kg, about 2.7-2.8 mg/kg, about 2.8-2.9 mg/kg, about 2.9-3.0mg/kg, about 3.0-3.1 mg/kg, about 3.1-3.2 mg/kg, about 3.2-3.3 mg/kg,about 3.3-3.4 mg/kg, about 3.4-3.5 mg/kg, about 3.5-3.6 mg/kg, about3.6-3.7 mg/kg, about 3.7-3.8 mg/kg, about 3.8-3.9 mg/kg, or about3.9-4.0 mg/kg).

The present disclosure also provides for the use of Compound 1 orCompound 1 in Form II, having a purity of equal to or greater than 91%wt/wt, e.g., having less than or equal to 9% wt/wt of other morphicforms or an amorphous form, in the manufacture of a medicament for thetherapeutic and/or prophylactic treatment of viral infection in asubject, e.g., an immunodeficient subject.

In another embodiment, the disclosure provides a method for thetherapeutic and/or prophylactic treatment of viral infection in asubject, e.g., an immunodeficient subject, the method comprisingadministering a compound of Formula II or III having a purity of equalto or greater than 91% wt/wt or being in Form II, e.g., having less thanor equal to 9% wt/wt of other morphic forms or an amorphous form, to thesubject.

In another embodiment, the disclosure also provides an oral dosage formcomprising a compound of Formula II or III having a purity of equal toor greater than about 91% wt/wt or being in Form II, e.g., having lessthan or equal to about 9% wt/wt of other morphic forms or an amorphousform, for the therapeutic and/or prophylactic treatment of viralinfection in a subject, wherein said oral dosage form, uponadministration to a human at a dosage of about 2 mg/kg of said compound,provides an AUC_(0-inf) of said compound of about 2000 to about 4000h*ng/mL, e.g., about 2500 to about 3000 h*ng/mL.

In another embodiment, the disclosure also provides an oral dosage formcomprising a compound of Formula II or III having a purity of equal toor greater than about 91% wt/wt or being in Form II, e.g., having lessthan or equal to about 9% wt/wt of other morphic forms or an amorphousform, for the therapeutic and/or prophylactic treatment of viralinfection in a subject, wherein said oral dosage form, uponadministration to a human at a dosage of about 1-2 mg/kg, about 2-3mg/kg, about 3-4 mg/kg of said compound, provides a C_(max) of saidcompound of about 100 to about 500 ng/mL, e.g., about 200 to about 400h*ng/mL.

In another embodiment, the disclosure also provides an oral dosage formcomprising a compound of Formula II or III having a purity of equal toor greater than about 91% wt/wt or being in Form II, e.g., having lessthan or equal to about 9% wt/wt of other morphic forms or an amorphousform, for the therapeutic and/or prophylactic treatment of viralinfection in a subject, wherein said oral dosage form, uponadministration to a human at a dosage of about 1-2 mg/kg, about 2-3mg/kg, about 3-4 mg/kg of said compound of Formula II or III andmetabolism of said compound of Formula II or III to cidofovir, providesa C_(max) of said cidofovir that is less than about 30% of the C_(max)of said compound of Formula II or III, e.g., less that about 20% of theC_(max) of said compound of Formula II or III.

Recrystallization Protocols

In one or more embodiments, the present technology includes fivedistrict recrystallization steps to purify Compound 1. For instance, insome preferred embodiments, the following schedule is used for thesequential recrystallization:

1st Recrystallization Dissolve in MeOH 60-70 C, stir 5 min. Cool to 60 Cand stir 1 h Cool to 50 C over 6 h Cool to 20 C over 2 h and stir 2 hFilter 2nd Recrystallization Dissolve in MeOH 60-70° C., stir 5 min.Cool to 60° C. and stir 1 h Cool to 50° C. over 6 h Cool to 20° C. over2 h and stir 2 h Filter 3rd Recrystallization Dissolve in MeOH at 60-70°C., stir 20 min. Cool to 60° C., stir 20 min., add seed stock, stir 2 hCool to 50° C. over 8 h and stir 2 h Cool to 20 over 6 h and stir 2 hFilter, dry ≦40 C, mill 4th Recrystallization Dissolve in MeOH 60-70°C., stir 20 min. Add n-heptane keeping above 50° C., stir 20 min. Coolto 40° C. over 6 h, stir 1 h Cool to 20° C. over 6 h, stir 2 h Filter5th Recrystallization Dissolve in MeOH at 60-70° C., stir 20 min. Coolto 61° C., add seed stock, stir 2 h Cool to 50° C. over 8 h, stir 2 hCool to 20° C. over 6 h, stir 2 h Filter, dry ≦40° C., mill

In some embodiments, other recrystallization schedules can be used. Forinstance, in some embodiments, Compound 1 can be subject to a singlemethanol recrystallization before recrystallizing with n-heptane andmethanol. This can then be followed by a final recrystallization withmethanol including seeding with Form II. Alternative schedules forrecrystallization and purification can be envisioned by one of skill inthe art, and the following illustrative embodiments are not to beconstrued as limiting:

Recrystallization Embodiment A

1st Recrystallization Dissolve in MeOH 60-70° C., stir 5 min. Cool to60° C. and stir 1 h Cool to 50° C. over 6 h Cool to 20° C. over 2 h andstir 2 h Filter 2nd Recrystallization Dissolve in MeOH 60-70° C., stir20 min. Add n-heptane keeping around 50 C, stir 20 min. Cool to 35° C.over 4 h, stir 1 h Cool to 20° C. over 6 h, stir 2 h Filter 3rdRecrystallization Dissolve in MeOH at 60-70° C., stir 20 min. Cool to61° C., add seed stock, stir 2 h Cool to 50° C. over 8 h, stir 2 h Coolto 20° C. over 6 h, stir 2 h Filter, dry ≦40° C., mill

Recrystallization Embodiment B

1st Recrystallization Dissolve in MeOH 60-70° C., stir 20 min. Addn-heptane keeping above 50° C., stir 20 min. Cool to 4° C. over 6 h,stir 1 h Cool to 20° C. over 6 h, stir 2 h Filter 2nd RecrystallizationDissolve in MeOH at 60-70° C., stir 20 min. Cool to 60° C., stir 20min., add seed stock, stir 2 h Cool to 50° C. over 8 h and stir 2 h Coolto 20° C. over 6 h and stir 2 h Filter, dry ≦40° C., mill 3rdRecrystallization Dissolve in MeOH at 60-70° C., stir 20 min. Cool to61° C., add seed stock, stir 2 h Cool to 50° C. over 8 h, stir 2 h Coolto 20° C. over 6 h, stir 2 h Filter, dry ≦40° C., mill

In some embodiments, alternative recrystallization protocols can beemployed which will be apparent to one of skill in the art. One of skillin the art will recognize that different crystallizations can be more orless effective at removing different types of impurities.

TABLE 8 Crystallization Experiments of a Compound having Formula II orIII in Methanol Conditions^(a) Observation Method Result ~9 vols.Unseeded. ~91% Crystallizatin occurred at XRPD Form II yield. ~54-56°C.; Aggregates, irregular plates, B/E ~8.5 vols; Seed ~2% hand Opaqueaggregates and XRPD Form II ground Compound 1 Form small plates, B/E SEMVery large II at ~61° C. Dissolved. agglomerates Seed with ~2% ground(>500 μm Compound 1 Form II at ~59° C. diameter); ~10 vols. Seed ~0.5%hand Aggregates and plates, B/E XRPD Form II ground Compound 1 Form IISEM Agglomerates at ~58° C. Hold ~1 hour. ~94% yield. (~500 μmdiameter); plates (up to ~100 μm)^(b) PSA d10 = 17.8 μm d50 = 69.6 μm;d90 = 160.9 μm ~10 vols. Seed ~3% hand Aggregates and plates, B/E XRPDForm II ground Compound 1 Form SEM Agglomerates II. Hold ~1hour. ~91%yield. (~200 μm diameter); plates (up to −50 μm)^(b) PSA d10 = 9.8 μm;d50 = 34.4 μm; d90 = 101.7 μm; ~8.5 vols. Unseeded. Hold ~4 h Aggregatesand plates, B/E XRPD Form II at ~58° C. (ppt). ~90% yield. ~10 vols.Unseeded. Hold ~4 h Aggregates and plates, B/E XRPD Form II at ~56° C.~90% yield. ~10 vols. Used Compound 1 Aggregates and plates, B/E XRPDForm II Form H as starting material. Seed ~0.5% hand ground Compound 1.Hold ~1 h. ~85% yields. ~10 vols. Used Compound 1 Aggregates and plates,B/E XRPD Form II Form H as starting material. SEM Large plates Unseeded.Hold ~4 h at ~56° C. (up to ~100 ~86% yield. ~10 vols in 97:3 Aggregatesand plates, B/E XRPD Form II Methanol:water. Unseeded. Hold ~4 h at ~56°C. ~92% yield. ~10 vols in 93:7 Aggregates and small XRPD Form IIMethanol:water. Unseeded. plates, B/E SEM Plates (up to Hold ~4 h at~56° C. ~94% ~100 μm)^(b) yield. ~10 vols. Slow agitation Aggregates andsmall XRPD Form II (~25 rpm). Unseeded. Hold plates, B/E SEMAgglomerates ~4 h at ~57° C. ~94% yield. (~200 μm diameter) and plates(up to −50 μm)^(b) ~10 vols. Slow agitation (~25 rpm). Aggregates andplates, B/E XRPD Form II Unseeded. Hold at ~57° C. SEM Large 3 h at slowagitation, 1 h agglomerates at high agitation (~350 rpm). (~500 μm Slowagitation for remaining diameter) and cool. Stir at ~15° C. at highplates (up to agitation prior to isolation. ~50 μm)^(b) ~95% yield.^(a)Crystallizations were conducted under non-GMP conditions usingEasyMax ™. Temperatures, times, and rates were approximate.Crystallizations occurred in methanol unless otherwise specified. Ratiosgiven are volumetric. All experiments used Compound 1 Form II sample asthe starting material unless otherwise specified. Slow cooling (9-10hours) was used in each of the crystallizations. Samples were dried in avacuum oven between ~40° C. and ~48° C. for 7 hours−1 day unlessotherwise specified. Scale was ~5 g. ^(b)Observation based on SEMimages. ^(c)Significant loss of solvent was observed in reactor likelydue to evaporation.

Pharmaceutical Compositions

In another aspect, provided herein is a pharmaceutical compositioncomprising polymorphs of the present invention (e.g., Polymorph FormII), and optionally a pharmaceutically acceptable carrier or diluent.Also provided herein is a pharmaceutical composition comprisingpolymorphs of the present invention (e.g., Polymorph or Form II) and apharmaceutically acceptable carrier or diluent.

The term “pharmaceutical composition” includes preparations suitable foradministration to mammals, e.g., humans. When the compounds of thepresent invention are administered as pharmaceuticals to mammals, e.g.,humans, they can be given per se or as a pharmaceutical compositioncontaining, for example, about 0.1% to about 99.9%, about 0.2 to about98%, about 0.3% to about 97%, about 0.4% to about 96%, or about 0.5 toabout 95% of active ingredient in combination with a pharmaceuticallyacceptable carrier. In one embodiment pharmaceutical compositioncontaining about 0.5% to about 90% of active ingredient in combinationwith a pharmaceutically acceptable carrier is suitable foradministration to mammals, e.g., humans. Some embodiments providepreparation of a pharmaceutical composition comprising about 0.1% toabout 99.9%, about 0.2 to about 98%, about 0.3% to about 97%, about 0.4%to about 96%, or about 0.5 to about 95% of the compound of Formula II orIII of the present invention for use in treating, preventing, orprophylaxis of viral infections or viral infection associated disorders.The present disclosure provides use of about 0.1% to about 99.9%, about0.2 to about 98%, about 0.3% to about 97%, about 0.4% to about 96%, orabout 0.5 to about 95% of the compound of Formula II or III for themanufacture of a medicament containing effective amounts of the compoundfor use in treating, preventing, or prophylaxis of viral infections andviral infection associated diseases.

In some embodiments, the pharmaceutical composition comprises ananhydrous morphic form (e.g., Form II) of the compound of Formula II orIII (or a pharmaceutically acceptable salt thereof), which issubstantially free of Form I and/or Form H. The pharmaceuticalcomposition comprising morphic Form II of the present disclosure hasequal to or less than about 10% Form I and/or Form H as impurities. Insome embodiments, the pharmaceutical composition comprising Form II hasequal to or less than about 9%, about 8%, about 7%, about 6%, about 5%,about 4%, about 3%, about 2%, about 1%, about 0.9%, about 0.8%, about0.7%, about 0.6%, about 0.5%, about 0.4%, about 0.3%, about 0.2%, about0.1%, about 0.05%, about 0.01%, or about 0.001% Form I and/or Form H asimpurities.

The polymorphs described herein (e.g., Polymorph II) may be combinedwith a pharmaceutically acceptable carrier according to conventionalpharmaceutical compounding techniques. As used herein, “pharmaceuticallyacceptable carrier” may include any and all solvents, diluents, or otherliquid vehicle, dispersion or suspension aids, surface active agents,isotonic agents, thickening or emulsifying agents, preservatives, solidbinders, lubricants and the like, as suited to the particular dosageform desired. Remington's Pharmaceutical Sciences, Sixteenth Edition, E.W. Martin (Mack Publishing Co., Easton, Pa., 1980) discloses variouscarriers used in formulating pharmaceutical compositions and knowntechniques for the preparation thereof. Except insofar as anyconventional carrier medium is incompatible with the compounds such asby producing any undesirable biological effect or otherwise interactingin a deleterious manner with any other component(s) of thepharmaceutical composition, its use is contemplated to be within thescope of this invention. Some examples of materials which can serve aspharmaceutically acceptable carriers include, but are not limited to,sugars such as lactose, glucose and sucrose; starches such as cornstarch and potato starch; cellulose and its derivatives such as sodiumcarboxymethyl cellulose, ethyl cellulose and cellulose acetate; powderedtragacanth; malt; gelatine; talc; excipients such as cocoa butter andsuppository waxes; oils such as peanut oil, cottonseed oil; saffloweroil, sesame oil; olive oil; corn oil and soybean oil; glycols; such aspropylene glycol; esters such as ethyl oleate and ethyl laurate; agar;buffering agents such as magnesium hydroxide and aluminum hydroxide;alginic acid; pyrogen free water; isotonic saline; Ringer's solution;ethyl alcohol, and phosphate buffer solutions, as well as othernon-toxic compatible lubricants such as sodium lauryl sulfate andmagnesium stearate, as well as coloring agents, releasing agents,coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the composition,according to the judgment of the formulator.

Furthermore, the carrier may take a wide variety of forms depending onthe form of the preparation desired for administration, e.g. oral,nasal, rectal, vaginal, parenteral (including intravenous injections orinfusions). In preparing compositions for oral dosage form any of theusual pharmaceutical media may be employed. Usual pharmaceutical mediainclude, for example, water, glycols, oils, alcohols, flavoring agents,preservatives, coloring agents, and the like in the case of oral liquidpreparations (such as for example, suspensions, solutions, emulsions andelixirs); aerosols; or carriers such as starches, sugars,microcrystalline cellulose, diluents, granulating agents, lubricants,binders, disintegrating agents and the like, in the case of oral solidpreparations (such as for example, powders, capsules, and tablets).

Pharmaceutical compositions comprising the polymorphs of the presentinvention (e.g., Form II) may be formulated to have any concentrationdesired. In some embodiments, the composition is formulated such that itcomprises at least a therapeutically effective amount. As used herein,“therapeutically effective amount” means that amount necessary to make aclinically observed improvement in the patient. In some embodiments, thecomposition is formulated such that it comprises an amount that wouldnot cause one or more unwanted side effects.

Pharmaceutical compositions include those suitable for oral, sublingual,nasal rectal, vaginal, topical, buccal and parenteral (includingsubcutaneous, intramuscular, and intravenous) administration, althoughthe most suitable route will depend on the nature and severity of thecondition being treated. The compositions may be conveniently presentedin unit dosage form, and prepared by any of the methods well known inthe art of pharmacy. In certain embodiments, the pharmaceuticalcomposition is formulated for oral administration in the form of a pill,capsule, lozenge or tablet. In other embodiments, the pharmaceuticalcomposition is in the form of a suspension.

The regimen of administration can affect what constitutes apharmaceutically effective amount. A polymorph of the present invention(e.g., Form II), and compositions thereof, can be administered to thesubject either prior to or after the onset of a disease. Further,several divided dosages, as well as staggered dosages can beadministered daily or sequentially, or the dose can be continuouslyinfused, or can be a bolus injection. Further, the dosages can beproportionally increased or decreased as indicated by the exigencies ofthe therapeutic or prophylactic situation. Further, the dosages may beco-administered in combination with other chemotherapeutic agents knownby the skilled artisan.

A “pharmaceutical composition” is a formulation containing a compound ofthe present invention in a form suitable for administration to asubject. In one embodiment, the pharmaceutical composition is in bulk orin unit dosage form. The unit dosage form is any of a variety of forms,including, for example, a capsule, an IV bag, a tablet, a single pump onan aerosol inhaler or a vial. The quantity of active ingredient (e.g., aformulation of the disclosed compound or salt, hydrate, solvate orisomer thereof) in a unit dose of composition is an effective amount andis varied according to the particular treatment involved. One skilled inthe art will appreciate that it is sometimes necessary to make routinevariations to the dosage depending on the age and condition of thepatient. The dosage will also depend on the route of administration. Avariety of routes are contemplated, including oral, pulmonary, rectal,parenteral, transdermal, subcutaneous, intravenous, intramuscular,intraperitoneal, inhalational, buccal, sublingual, intrapleural,intrathecal, intranasal, and the like. Dosage forms for the topical ortransdermal administration of a compound of this invention includepowders, sprays, ointments, pastes, creams, lotions, gels, solutions,patches and inhalants. In one embodiment, the active compound is mixedunder sterile conditions with a pharmaceutically acceptable carrier, andwith any preservatives, buffers or propellants that are required.

As used herein, the phrase “pharmaceutically acceptable” refers to thosecompounds, materials, compositions, carriers, and/or dosage forms whichare, within the scope of sound medical judgment, suitable for use incontact with the tissues of human beings and animals without excessivetoxicity, irritation, allergic response, or other problem orcomplication, commensurate with a reasonable benefit/risk ratio.

“Pharmaceutically acceptable excipient or carrier” means an excipient orcarrier that is useful in preparing a pharmaceutical composition that isgenerally safe, non-toxic and neither biologically nor otherwiseundesirable, and includes excipient that is acceptable for veterinaryuse as well as human pharmaceutical use. A “pharmaceutically acceptableexcipient” as used in the specification and claims includes both one andmore than one such excipient.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (topical), andtransmucosal administration. Solutions or suspensions used forparenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates, and agents for theadjustment of tonicity such as sodium chloride or dextrose. The pH canbe adjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

The term “therapeutically effective amount”, as used herein, refers toan amount of a pharmaceutical agent to treat, ameliorate, or prevent anidentified disease or condition, or to exhibit a detectable therapeuticor inhibitory effect. The effect can be detected by any assay methodknown in the art. The precise effective amount for a subject will dependupon the subject's body weight, size, and health; the nature and extentof the condition; and the therapeutic or combination of therapeuticsselected for administration. Therapeutically effective amounts for agiven situation can be determined by routine experimentation that iswithin the skill and judgment of the clinician. In a preferred aspect,the disease or condition to be treated is viral infection.

For any compound, the therapeutically effective amount can be estimatedinitially either in cell culture assays, e.g., of neoplastic cells, orin animal models, usually rats, mice, rabbits, dogs, or pigs. The animalmodel may also be used to determine the appropriate concentration rangeand route of administration. Such information can then be used todetermine useful doses and routes for administration in humans.Therapeutic/prophylactic efficacy and toxicity may be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., ED₅₀ (the dose therapeutically effective in 50% of thepopulation) and LD₅₀ (the dose lethal to 50% of the population). Thedose ratio between toxic and therapeutic effects is the therapeuticindex, and it can be expressed as the ratio, LD₅₀/ED₅₀. Pharmaceuticalcompositions that exhibit large therapeutic indices are preferred. Thedosage may vary within this range depending upon the dosage formemployed, sensitivity of the patient, and the route of administration.

Dosage and administration are adjusted to provide sufficient levels ofthe active agent(s) or to maintain the desired effect. Factors which maybe taken into account include the severity of the disease state, generalhealth of the subject, age, weight, and gender of the subject, diet,time and frequency of administration, drug combination(s), reactionsensitivities, and tolerance/response to therapy. Long-actingpharmaceutical compositions may be administered every 3 to 4 days, everyweek, or once every two weeks depending on half-life and clearance rateof the particular formulation.

The pharmaceutical compositions containing active compounds of thepresent invention may be manufactured in a manner that is generallyknown, e.g., by means of conventional mixing, dissolving, granulating,dragee-making, levigating, emulsifying, encapsulating, entrapping, orlyophilizing processes. Pharmaceutical compositions may be formulated ina conventional manner using one or more pharmaceutically acceptablecarriers comprising excipients and/or auxiliaries that facilitateprocessing of the active compounds into preparations that can be usedpharmaceutically. Of course, the appropriate formulation is dependentupon the route of administration chosen.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringeability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, methods of preparation are vacuum dryingand freeze-drying that yields a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

Oral compositions generally include an inert diluent or an ediblepharmaceutically acceptable carrier. They can be enclosed in gelatincapsules or compressed into tablets. For the purpose of oral therapeuticadministration, the active compound can be incorporated with excipientsand used in the form of tablets, troches, or capsules. Oral compositionscan also be prepared using a fluid carrier for use as a mouthwash,wherein the compound in the fluid carrier is applied orally and swishedand expectorated or swallowed. Pharmaceutically compatible bindingagents, and/or adjuvant materials can be included as part of thecomposition. The tablets, pills, capsules, troches and the like cancontain any of the following ingredients, or compounds of a similarnature: a binder such as microcrystalline cellulose, gum tragacanth orgelatin; an excipient such as starch or lactose, a disintegrating agentsuch as alginic acid, Primogel, or corn starch; a lubricant such asmagnesium stearate or Sterotes; a glidant such as colloidal silicondioxide; a sweetening agent such as sucrose or saccharin; or a flavoringagent such as peppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser, whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The active compounds can be prepared with pharmaceutically acceptablecarriers that will protect the compound against rapid elimination fromthe body, such as a controlled release formulation, including implantsand microencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved.

Pharmaceutical compositions comprising Form II of a compound havingFormula II or III (or a pharmaceutically acceptable salt thereof) can beidentified by comparison of the compositions' X-ray powder diffractionpatterns to an X-ray powder diffraction pattern of Form II. It will beappreciated that pharmaceutical compositions comprising Form II of acompound having Formula II or III (or a pharmaceutically acceptable saltthereof) exhibit non-identical X-ray powder diffraction patterns thatare substantially the same pattern as compared to FIG. 1. Observedslight differences in XRPD patterns may be attributed to theaforementioned factors, including the presence of other impurities inthe sample.

The tablets were lightly ground using a mortar and pestle prior toanalysis. The tablets show very similar XRPD patterns similar to FIG. 13and/or FIG. 14, indicative of crystalline material with diffuse scatterpotentially from one or more excipients.

In one embodiment, Form II of a compound having Formula II or III (or apharmaceutically acceptable salt thereof) exhibits an X-ray powderdiffraction pattern having a characteristic peaks expressed in degrees2-theta (±0.2) at 2.81, 5.63, 19.00, 19.57, 22.76, and 24.70, or havingan X-ray diffraction pattern substantially similar to that set forth inFIG. 13 or 17, indexing substantially similar to that set forth in FIG.2, and DSC Thermogram substantially similar to that set forth in FIG. 15or 16.

In some embodiments, the DSC thermograms for two Form II tablet samplesshow overlapping minor endotherms at about 90° C. and about 95° C. (peakmax) and a major endotherm with an onset at about 165° C. The tabletsamples have a large endotherm at about 196° C. of the Form IIcrystalline form.

In one embodiment, hot stage microscopy does not show any significantobservations prior to flow at about 189° C. other than some potentialsublimation observed at about 98° C. In some embodiments, the Form II ofthe present embodiments has a potential thermotropic mesophase.

TABLE 9 Characterization of Form II Tablet 1 Sample MethodAnalysis/Result FIG. Form II DSC Minor overlapping endos at ~90° C. 18Tablet-1 and ~95° C.; Major endo XRPD Crystalline with diffuse scatter;16 Peaks consistent with Form II ^(a)Sample was submitted as whitetablet but was lightly hand ground in mortar and pestle for analysis

TABLE 10 Characterization of Form II Tablet 2 Sample MethodAnalysis/Result FIG. Form II DSC Minor overlapping endos at ~91° C. 19Tablet-2 and ~95° C.; Major endo XRPD Crystalline with diffuse scatter;17 Peaks consistent with Form II ^(a)Sample was submitted as whitetablet but was lightly hand ground in mortar and pestle for analysis

In therapeutic applications, the dosages of the pharmaceuticalcompositions used in accordance with the invention vary depending on theagent, the age, weight, and clinical condition of the recipient patient,and the experience and judgment of the clinician or practitioneradministering the therapy, among other factors affecting the selecteddosage. Dosages can range from about 0.01 mg/kg to about 100 mg/kg. Inpreferred aspects, dosages can range from about 0.1 mg/kg to about 10mg/kg. In an aspect, the dose will be in the range of about 1 mg toabout 1 g; about 10 mg to about 500 mg; about 20 mg to about 400 mg;about 40 mg to about 400 mg; or about 50 mg to about 400 mg, in single,divided, or continuous doses (which dose may be adjusted for thepatient's weight in kg, body surface area in m², and age in years). Incertain embodiments, the amount per dosage form can be about 0.1 mg toabout 1000 mg, e.g., about 0.1 mg, about 0.5 mg, about 1.0 mg, about 2.0mg, about 3.0 mg, about 4.0 mg, about 5.0 mg, about 6.0 mg, about 7.0mg, about 8.0 mg, about 9.0 mg, about 10 mg, about 15 mg, about 20 mg,about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, about50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, about 75 mg,about 80 mg, about 85 mg, about 90 mg, about 95 mg, or about 100 mg ormore. In one embodiment, the amount can be about 20 mg. In oneembodiment, the amount can be about 50 mg.

Form II or Form H of Compound 1 or pharmaceutically acceptable saltsthereof is formulated as a pharmaceutical composition or is used in themanufacture of a medicament for the treatment of a viral infectionand/or viral infection associated disease and/or disorder. Thecomposition and/or the medicament of Form II or Form H of Compound 1 orpharmaceutically acceptable salts thereof is formulated as a tablet orsuspension. Tablets of Compound 1 Form II is formulated comprisingpharmacologically acceptable buffers, excipients, carriers, includingemulsifiers, enhancers (e.g., absorption enhancers), disintegrants(e.g., Polyvinylpolypyrrolidone (polyvinyl polypyrrolidone, PVPP,crospovidone, crospolividone or E1202), which is a highly cross-linkedmodification of polyvinylpyrrolidone (PVP)), and/or polymers disclosedin the present disclosure and well-known in the art.

In one embodiment, the present disclosure provides Tablet Formulation 1of Compound 1 for use in prophylactic treatment or prevention viralinfection and/or viral associated disease or disorder. The presentdisclosure provides Tablet Formulation 1 of Compound 1 for use intreating immunodeficient subjects, or pre- or post-organ and/or tissuetransplantation subjects.

In another embodiment, the present disclosure provides TabletFormulation 2 of Compound 1 for use in prophylactic treatment orprevention viral infection and/or viral associated disease and/ordisorder. The present disclosure provides Tablet Formulation 2 ofCompound 1 for use in treating immunodeficient subjects, or pre- orpost-organ and/or tissue transplantation subjects.

In some embodiments, the present disclosure provides Tablet Formulation1 of Compound 1 Form II for use in prophylactic treatment or preventionviral infection and/or viral associated disease and/or disorder. Thepresent disclosure provides Tablet Formulation 1 of Compound 1 Form IIfor use in treating immunodeficient subjects, or pre- or post-organand/or tissue transplantation subjects.

In some embodiments, the present disclosure provides Tablet Formulation2 of Compound 1 Form II for use in prophylactic treatment or preventionviral infection and/or viral associated disease and/or disorder. Thepresent disclosure provides Tablet Formulation 2 of Compound 1 Form IIfor use in treating immunodeficient subjects, or pre- or post-organand/or tissue transplantation subjects.

Compositions of two Compound 1 formulations in tablet form of thecurrent disclosure are listed in Table 11.

In one embodiment, the present disclosure provides suspensionFormulation 3 of Compound 1 for use in prophylactic treatment orprevention viral infection and/or viral associated disease and/ordisorder. The present disclosure provides suspension Formulation 3 ofCompound 1 for use in treating immunodeficient subjects, or pre- orpost-organ and/or tissue transplantation subjects.

In another embodiment, the present disclosure provides suspensionFormulation 4 of Compound 1 for use in prophylactic treatment orprevention viral infection and/or viral associated disease and/ordisorder. The present disclosure provides suspension Formulation 4 ofCompound 1 for use in treating immunodeficient subjects, or pre- orpost-organ and/or tissue transplantation subjects.

TABLE 11 100 mg Tablet formulations of Compound 1 Tablet FormulationsFormulation 1 Formulation 2 % wt per % wt per Ingredient mg/tablettablet mg/tablet tablet Compound 1 100.00 27.78 100.00 27.8 Silicified79.86 22.18 80.0 22.2 Microcrystalline Cellulose (Prosolv 90),Crospovidone 13.37 3.714 13.4 3.7 (Polyplasdone XL-10) Microcrystalline40.93 11.37 —/— —/— Cellulose and Mannitol (Avicel HFE 102)Microcrystalline —/— —/— 41.0 11.4 Cellulose (Avicel PHE102) Mannitol124.10 34.46 122.0 33.9 (Pearlitol 100 SD) Colloidal Silicon Dioxide —/——/— 1.8 0.5 (Cab-O-Sil) Magnesium Stearate 0.5 Total 360 100% 360 100%

In some embodiments, the present disclosure provides suspensionFormulation 3 or 4 of Compound 1 Form II for use in prophylactictreatment and/or prevention viral infection and/or viral associateddisease and/or disorder. The present disclosure provides suspensionFormulation 3 or 4 of Compound 1 Form II for use in treatingimmunodeficient subjects, or pre- or post-organ and/or tissuetransplantation subjects.

Compositions of two Compound 1 formulations in suspension form of thecurrent disclosure are listed in Table 12.

TABLE 12 Suspension Formulations of Compound 1 Suspension FormulationsFormulation 3 (in situ Formulation 4 precipitation) (direct wetting)Ingredient g/L % wt g/L % wt Compound 1 10.00 0.907 100.00 1.00 SodiumPhosphate, dibasic 0.650 0.059 —/— —/— Citric Acid, monohydrate 1.5000.136 0.585 0.06 Sodium Citrate —/— —/— 0.985 0.10 Xantham Gum 1.2500.113 0.375 0.04 Methylparaben, sodium salt 0.850 0.077 1.690 0.17Propylparaben, sodium salt 0.085 0.0077 0.190 0.02 Sucralose 0.200 0.0180.500 0.05 Microcrystalline Cellulose and 15.00 1.360 15.625 1.56Carboxymethylcellulose Sodium (VivaPur MCG 591) High Fructose Corn Syrup426.6 38.68 276.720 27.67 (55%) Lemon Lime Flavor 1.500 0.136 4.000 0.40(WONF220J15) Sodium Hydroxide, pellets 0.700 0.0635 Purified Water 644.558.43 689.335 68.93 Sodium qs qs qs qs Hydroxide/Hydrochloric Acid Total360 100% 360 100%

The formulations of the present disclosure are used in treatingend-organ damage related to viral infection, e.g. treating, preventing,and/or ameliorating BK virus infection associated end organ damage in asubject.

The formulations of the present disclosure are used in manufacturing amedicament in prophylactic treatment and/or prevention viral infectionand/or viral associated disease and/or disorder.

In one embodiment, Form II of a compound of Formula II or III (or apharmaceutically acceptable salt thereof) is administered at a dose ofabout 100 mg (tablet Formulation 1 or 2 described in Table 11, orsuspension Formulation 3 or 4 described in Table 12) twice a week. Inanother embodiment, Form II of a compound of Formula II or III (or apharmaceutically acceptable salt thereof) is administered at a dose ofabout 200 mg (tablet Formulation 1 or 2 described in Table 11, orsuspension Formulation 3 or 4 described in Table 12) once a week.

In another embodiment, the invention provides compositions (e.g.,pharmaceutical compositions) with desirable pharmacokineticcharacteristics. For example, the compositions of the invention mayprovide a blood level of Form II of a compound of Formula II or III (ora pharmaceutically acceptable salt thereof), which, after metabolism tothe therapeutically-active form (i.e., cidofovir), results in bloodlevels of the metabolite that do not induce toxicity (e.g.,nephrotoxicity).

An effective amount of a pharmaceutical agent is that which provides anobjectively identifiable improvement as noted by the clinician or otherqualified observer. As used herein, the term “dosage effective manner”refers to amount of an active compound to produce the desired biologicaleffect in a subject or cell.

In another embodiment, compound of Formula II, III, or anothercomposition of the present invention can be administered to a subject asa single dose. In another embodiment, Formula II, III, or anothercomposition of the present invention can be administered to a subject inmultiple doses. Multiple doses can be administered regularly, forexample, once every 12 hours, once a day, every 2 days, every 3 days,every 4 days, every 5 days, every 6 days, every 7 days, every 8 days,every 9 days, every 10 days, every 11 days, every 12 days, every 13days, every 14 days or every 15 days. For example, doses can beadministered twice per week. Moreover, each individual dose can beadministered with the same or a different dosage.

For example, a subject can be administered with a first dose of about1-4 mg/kg (e.g., about 1-1.1 mg/kg, about 1.1-1.2 mg/kg, about 1.2-1.3mg/kg, about 1.3-1.4 mg/kg, about 1.4-1.5 mg/kg, about 1.5-1.6 mg/kg,about 1.6-1.7 mg/kg, about 1.7-1.8 mg/kg, about 1.8-1.9 mg/kg, about1.9-2.0 mg/kg, about 2.0-2.1 mg/kg, about 2.1-2.2 mg/kg, about 2.2-2.3mg/kg, about 2.3-2.4 mg/kg, about 2.4-2.5 mg/kg, about 2.5-2.6 mg/kg,about 2.6-2.7 mg/kg, about 2.7-2.8 mg/kg, about 2.8-2.9 mg/kg, about2.9-3.0 mg/kg, about 3.0-3.1 mg/kg, about 3.1-3.2 mg/kg, about 3.2-3.3mg/kg, about 3.3-3.4 mg/kg, about 3.4-3.5 mg/kg, about 3.5-3.6 mg/kg,about 3.6-3.7 mg/kg, about 3.7-3.8 mg/kg, about 3.8-3.9 mg/kg, or about3.9-4.0 mg/kg) of Form II of a compound having Formula II or III (or apharmaceutically acceptable salt thereof) followed by one or moreadditional doses at 1-4 mg/kg (e.g., about 1-1.1 mg/kg, about 1.1-1.2mg/kg, about 1.2-1.3 mg/kg, about 1.3-1.4 mg/kg, about 1.4-1.5 mg/kg,about 1.5-1.6 mg/kg, about 1.6-1.7 mg/kg, about 1.7-1.8 mg/kg, about1.8-1.9 mg/kg, about 1.9-2.0 mg/kg, about 2.0-2.1 mg/kg, about 2.1-2.2mg/kg, about 2.2-2.3 mg/kg, about 2.3-2.4 mg/kg, about 2.4-2.5 mg/kg,about 2.5-2.6 mg/kg, about 2.6-2.7 mg/kg, about 2.7-2.8 mg/kg, about2.8-2.9 mg/kg, about 2.9-3.0 mg/kg, about 3.0-3.1 mg/kg, about 3.1-3.2mg/kg, about 3.2-3.3 mg/kg, about 3.3-3.4 mg/kg, about 3.4-3.5 mg/kg,about 3.5-3.6 mg/kg, about 3.6-3.7 mg/kg, about 3.7-3.8 mg/kg, about3.8-3.9 mg/kg, or about 3.9-4.0 mg/kg) of Form II of Formula II or III(or a pharmaceutically acceptable salt thereof) in the same week or inthe following week. For example, a subject can be administered with afirst dose of about 3 mg/kg followed by one or more additional doses atabout 1 mg/kg. For example, a subject can be administered with a firstdose of about 2 mg/kg followed by one or more additional doses at about3 mg/kg. For example, a subject can be administered with a first dose of4 mg/kg followed by one or more additional doses at about 4 mg/kg.

Multiple doses can also be administered at variable time intervals. Forexample, the first 2, 3, 4, 5, 6, 7, or 8 or more doses can beadministered at an interval of 6 days followed by additional dosesadministered at an interval of 7 days. For example, the first 2, 3, 4,5, 6, 7, or 8 or more doses can be administered at an interval of 7 daysfollowed by additional doses administered at an interval of 3 days.

In another embodiment, the invention provides an oral dosage formcomprising Form II of a compound having Formula II or III (or apharmaceutically acceptable salt thereof) having a purity of greaterthan 91% or being in Form II for the therapeutic and/or prophylactictreatment of viral infection in a subject, wherein said oral dosageform, upon administration to a human at a dosage of about 1-4 mg/kg(e.g., about 1-1.1 mg/kg, about 1.1-1.2 mg/kg, about 1.2-1.3 mg/kg,about 1.3-1.4 mg/kg, about 1.4-1.5 mg/kg, about 1.5-1.6 mg/kg, about1.6-1.7 mg/kg, about 1.7-1.8 mg/kg, about 1.8-1.9 mg/kg, about 1.9-2.0mg/kg, about 2.0-2.1 mg/kg, about 2.1-2.2 mg/kg, about 2.2-2.3 mg/kg,about 2.3-2.4 mg/kg, about 2.4-2.5 mg/kg, about 2.5-2.6 mg/kg, about2.6-2.7 mg/kg, about 2.7-2.8 mg/kg, about 2.8-2.9 mg/kg, about 2.9-3.0mg/kg, about 3.0-3.1 mg/kg, about 3.1-3.2 mg/kg, about 3.2-3.3 mg/kg,about 3.3-3.4 mg/kg, about 3.4-3.5 mg/kg, about 3.5-3.6 mg/kg, about3.6-3.7 mg/kg, about 3.7-3.8 mg/kg, about 3.8-3.9 mg/kg, or about3.9-4.0 mg/kg) of said compound, provides an AUC_(0-inf) of saidcompound of about 2000 to about 4000 h*ng/mL, e.g., about 2500 to about3000 h*ng/mL. In some embodiments, the AUC_(0-inf) of said compound isabout 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000,3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, or 4000 h*ng/mL orany range therein. AUC_(0-inf) can be determined by any of thewell-known methods in the art and as described in the examples herein.

In another embodiment, the invention provides an oral dosage formcomprising Form II of a compound of Formula II or III (or apharmaceutically acceptable salt thereof) having a purity of equal to orgreater than 91% or being in Form II for the therapeutic and/orprophylactic treatment of viral infection in a subject, wherein saidoral dosage form, upon administration to a human at a dosage of about1-4 mg/kg (e.g., about 1-1.1 mg/kg, about 1.1-1.2 mg/kg, about 1.2-1.3mg/kg, about 1.3-1.4 mg/kg, about 1.4-1.5 mg/kg, about 1.5-1.6 mg/kg,about 1.6-1.7 mg/kg, about 1.7-1.8 mg/kg, about 1.8-1.9 mg/kg, about1.9-2.0 mg/kg, about 2.0-2.1 mg/kg, about 2.1-2.2 mg/kg, about 2.2-2.3mg/kg, about 2.3-2.4 mg/kg, about 2.4-2.5 mg/kg, about 2.5-2.6 mg/kg,about 2.6-2.7 mg/kg, about 2.7-2.8 mg/kg, about 2.8-2.9 mg/kg, about2.9-3.0 mg/kg, about 3.0-3.1 mg/kg, about 3.1-3.2 mg/kg, about 3.2-3.3mg/kg, about 3.3-3.4 mg/kg, about 3.4-3.5 mg/kg, about 3.5-3.6 mg/kg,about 3.6-3.7 mg/kg, about 3.7-3.8 mg/kg, about 3.8-3.9 mg/kg, or about3.9-4.0 mg/kg) of said compound, provides a C_(max) of said compound ofabout 100 to about 500 ng/mL, e.g., about 200 to about 400 ng/mL. Insome embodiments, the C_(max) of the compound is about 100, about 110,about 120, about 130, about 140, about 150, about 160, about 170, about180, about 190, about 200, about 210, about 220, about 230, about 240,about 250, about 260, about 270, about 280, about 290, about 300, about310, about 320, about 330, about 340, about 350, about 360, about 370,about 380, about 390, about 400, about 410, about 420, about 430, about440, about 450, about 460, about 470, about 480, about 490, or about 500ng/mL or any range therein. C_(max) can be determined by any of thewell-known methods in the art and as described in the examples herein.

In another embodiment, the invention provides an oral dosage formcomprising Form II of a compound having Formula II or III (or apharmaceutically acceptable salt thereof) having a purity of greaterthan about 91% or being in Form II for the therapeutic and/orprophylactic treatment of viral infection in a subject, wherein saidoral dosage form, upon administration to a human at a dosage of about1-4 mg/kg (e.g., about 1-1.1 mg/kg, about 1.1-1.2 mg/kg, about 1.2-1.3mg/kg, about 1.3-1.4 mg/kg, about 1.4-1.5 mg/kg, about 1.5-1.6 mg/kg,about 1.6-1.7 mg/kg, about 1.7-1.8 mg/kg, about 1.8-1.9 mg/kg, about1.9-2.0 mg/kg, about 2.0-2.1 mg/kg, about 2.1-2.2 mg/kg, about 2.2-2.3mg/kg, about 2.3-2.4 mg/kg, about 2.4-2.5 mg/kg, about 2.5-2.6 mg/kg,about 2.6-2.7 mg/kg, about 2.7-2.8 mg/kg, about 2.8-2.9 mg/kg, about2.9-3.0 mg/kg, about 3.0-3.1 mg/kg, about 3.1-3.2 mg/kg, about 3.2-3.3mg/kg, about 3.3-3.4 mg/kg, about 3.4-3.5 mg/kg, about 3.5-3.6 mg/kg,about 3.6-3.7 mg/kg, about 3.7-3.8 mg/kg, about 3.8-3.9 mg/kg, or about3.9-4.0 mg/kg) of said compound having Formula II or III (or apharmaceutically acceptable salt thereof) and metabolism of saidcompound of Formula II or III (or a pharmaceutically acceptable saltthereof) to cidofovir, provides a C_(max) of said cidofovir that is lessthan about 30% of the C_(max) of said compound having Formula II or III(or a pharmaceutically acceptable salt thereof), e.g., less that about20% of the C_(max) of said compound having Formula II or III (or apharmaceutically acceptable salt thereof). In some embodiments, theC_(max) of the metabolite (i.e., cidofovir) is less than about 50%, 45%,40%, 35%, 30%, 25%, 20%, 15%, or 10% of the C_(max) of Form II of acompound having Formula II or III (or a pharmaceutically acceptable saltthereof).

In another embodiment, the invention provides an oral dosage formcomprising a Form II of a compound having Formula II or III (or apharmaceutically acceptable salt thereof) having a purity of greaterthan 91% or being in Form II, wherein upon administration to a human ata dosage of about 2 mg/kg of said compound of Form II of a compoundhaving Formula II or III (or a pharmaceutically acceptable saltthereof), provides an AUC_(0-inf) of cidofovir of about 1000 to about5000 h*ng/mL, e.g., about 1500 to about 4000 h*ng/mL. In someembodiments, the AUC_(0-inf) of cidofovir is about 1000, 1100, 1200,1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400,2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600,3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800,4900, or 5000 h*ng/mL or any range therein.

In another embodiment, the invention provides an oral dosage formcomprising a Form II of a compound having Formula II or III (or apharmaceutically acceptable salt thereof) having a purity of equal to orgreater than 91% or being in Form II, wherein upon administration to ahuman at a dosage of about 2 mg/kg of said Form II of a compound havingFormula II or III (or a pharmaceutically acceptable salt thereof),provides a C_(max) of cidofovir of about 10 to about 100 ng/mL, e.g.,about 20 to about 70 ng/mL. In some embodiments, the C_(max) of Form IIof a compound having Formula II or III (or a pharmaceutically acceptablesalt thereof) is about 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 ng/mLor any range therein.

In certain embodiments, the oral dosage form provides more than one ofthe pharmacokinetic characteristics described above, e.g., theAUC_(0-inf) or C_(max) of Form II of a compound having Formula II or III(or a pharmaceutically acceptable salt thereof) or the metabolite (i.e.,cidofovir) or the C_(max) ratio of the metabolite (i.e., cidofovir) tothe compound of formula (I), e.g., 2, 3, 4, or more of thepharmacokinetic characteristics in any combination.

The pharmacokinetic behavior of a composition will vary somewhat fromsubject to subject within a population. The numbers described above forthe compositions of the invention are based on the average behavior in apopulation. The present invention is intended to encompass compositionsthat on average fall within the disclosed ranges, even though it isunderstood that certain subjects may fall outside of the ranges.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

The compounds of the present invention are capable of further formingsalts. All of these forms are also contemplated within the scope of theclaimed invention.

DEFINITIONS

As used herein, “pharmaceutically acceptable salts” refer to derivativesof the compounds of the present invention wherein the parent compound ismodified by making acid or base salts thereof. Examples ofpharmaceutically acceptable salts include, but are not limited to,mineral or organic acid salts of basic residues such as amines, alkalior organic salts of acidic residues such as carboxylic acids, and thelike. The pharmaceutically acceptable salts include the conventionalnon-toxic salts or the quaternary ammonium salts of the parent compoundformed, for example, from non-toxic inorganic or organic acids. Forexample, such conventional non-toxic salts include, but are not limitedto, those derived from inorganic and organic acids selected from2-acetoxybenzoic, 2-hydroxyethane sulfonic, acetic, ascorbic, benzenesulfonic, benzoic, bicarbonic, carbonic, citric, edetic, ethanedisulfonic, 1,2-ethane sulfonic, fumaric, glucoheptonic, gluconic,glutamic, glycolic, glycollyarsanilic, hexylresorcinic, hydrabamic,hydrobromic, hydrochloric, hydroiodic, hydroxymaleic, hydroxynaphthoic,isethionic, lactic, lactobionic, lauryl sulfonic, maleic, malic,mandelic, methane sulfonic, napsylic, nitric, oxalic, pamoic,pantothenic, phenylacetic, phosphoric, polygalacturonic, propionic,salicyclic, stearic, subacetic, succinic, sulfamic, sulfanilic,sulfuric, tannic, tartaric, toluene sulfonic, and the commonly occurringamine acids, e.g., glycine, alanine, phenylalanine, arginine, etc.

Other examples of pharmaceutically acceptable salts include hexanoicacid, cyclopentane propionic acid, pyruvic acid, malonic acid,3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, 4-chlorobenzenesulfonicacid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid,camphorsulfonic acid, 4-methylbicyclo-[2.2.2]-oct-2-ene-1-carboxylicacid, 3-phenylpropionic acid, trimethylacetic acid, tertiary butylaceticacid, muconic acid, and the like. The present invention also encompassessalts formed when an acidic proton present in the parent compound eitheris replaced by a metal ion, e.g., an alkali metal ion, an alkaline earthion, or an aluminum ion; or coordinates with an organic base such asethanolamine, diethanolamine, triethanolamine, tromethamine,N-methylglucamine, diethylamine, diethylaminoethanol, ethylenediamine,imidazole, lysine, arginine, morpholine, 2-hydroxyethylmorpholine,dibenzylethylenediamine, trimethylamine, piperidine, pyrrolidine,benzylamine, tetramethylammonium hydroxide and the like.

It should be understood that all references to pharmaceuticallyacceptable salts include solvent addition forms (solvates) or crystalforms (polymorphs) as defined herein, of the same salt.

The compounds of the present invention can also be prepared as esters,for example, pharmaceutically acceptable esters. For example, acarboxylic acid function group in a compound can be converted to itscorresponding ester, e.g., a methyl, ethyl or other ester. Also, analcohol group in a compound can be converted to its corresponding ester,e.g., acetate, propionate, or other esters.

The compounds, or pharmaceutically acceptable salts, esters orderivatives thereof, are administered orally, nasally, transdermally,pulmonary, inhalationally, buccally, sublingually, intraperintoneally,subcutaneously, intramuscularly, intravenously, rectally,intrapleurally, intrathecally and parenterally. In one embodiment, thecompound is administered orally. One skilled in the art will recognizethe advantages of certain routes of administration.

The dosage regimen utilizing the compounds is selected in accordancewith a variety of factors including type, species, age, weight, sex andmedical condition of the patient; the severity of the condition to betreated; the route of administration; the renal and hepatic function ofthe patient; and the particular compound or salt thereof employed. Anordinarily skilled physician or veterinarian can readily determine andprescribe the effective amount of the drug required to prevent, counteror arrest the progress of the condition.

Techniques for formulation and administration of the disclosed compoundsof the invention can be found in Remington: the Science and Practice ofPharmacy, 19^(th) edition, Mack Publishing Co., Easton, Pa. (1995). Inan embodiment, the compounds described herein, and the pharmaceuticallyacceptable salts thereof, are used in pharmaceutical preparations incombination with a pharmaceutically acceptable carrier or diluent.Suitable pharmaceutically acceptable carriers include inert solidfillers or diluents and sterile aqueous or organic solutions. Thecompounds will be present in such pharmaceutical compositions in amountssufficient to provide the desired dosage amount in the range describedherein.

It will be appreciated that the methods disclosed herein are suitablefor both large-scale and small-scale preparations of the desiredcompounds. In preferred embodiments of the methods described herein, thephosphonate esters may be prepared on a large scale, for example on anindustrial production scale rather than on an experimental/laboratoryscale. For example, a batch-type process according to the methods of thedisclosure allows the preparation of batches of at least 1 g, or atleast 5 g, or at least 10 g, or at least 100 g, or at least 1 kg, or atleast 100 kg of phosphonate ester product. Furthermore, the methodsallow the preparation of a phosphonate ester product having a purity ofat least 98%, or at least 98.5% as measured by HPLC. In preferredembodiments according to the disclosure, these products are obtained ina reaction sequence that does not involve purification by any form ofchromatography (e.g., gas chromatography, HPLC, preparative LC, sizeexclusion chromatography, and the like).

All patents, patent applications, and publications mentioned herein arehereby incorporated by reference in their entireties. However, where apatent, patent application, or publication containing expressdefinitions is incorporated by reference, those express definitionsshould be understood to apply to the incorporated patent, patentapplication, or publication in which they are found, and not to theremainder of the text of this application, in particular the claims ofthis application.

As used herein, a “subject” is interchangeable with a “subject in needthereof”, both of which refer to a subject having a disorder in whichviral infection plays a part, or a subject having an increased risk ofdeveloping viral infection associated disease or disorder relative tothe population at large. A “subject” includes a mammal. The mammal canbe e.g., a human or appropriate non-human mammal, such as primate,mouse, rat, dog, cat, cow, horse, goat, camel, sheep or a pig. Thesubject can also be a bird or fowl. In one embodiment, the mammal is ahuman.

As used herein, “treating,” “treatment” or “treat” describes themanagement and care of a patient for the purpose of combating a disease,condition, or disorder and includes the administration of a polymorph ofthe present invention (e.g., Polymorph II), to alleviate the symptoms orcomplications of a disease, condition or disorder, or to eliminate thedisease, condition or disorder. The term “treat” can also includetreatment of a cell in vitro or an animal model.

A polymorph of the present invention may also be used to prevent arelevant disease, condition or disorder, or used to identify suitablecandidates for such purposes. As used herein, “preventing,” “prevent,”or “protecting against” describes reducing, ameliorating or eliminatingthe onset of the symptoms or complications of such disease, condition ordisorder.

It is to be understood that while the invention has been described inconjunction with the preferred specific embodiments thereof, that theforegoing description as well as the examples that follow, are intendedto illustrate and not limit the scope of the invention. It will beunderstood by those skilled in the art that various changes may be madeand equivalents may be substituted without departing from the scope ofthe invention, and further that other aspects, advantages andmodifications will be apparent to those skilled in the art to which theinvention pertains.

As used herein “crystalline” means that the compound is crystallizedinto a specific crystal packing arrangement in three spatial dimensionsor the compound having external face planes. Compounds in thecrystalline state exhibit distinct sharp peaks in their X-raydiffraction patterns and typically exhibit well defined melting points.Different crystal forms usually have different X-ray diffractionpatterns, infrared spectral, melting points, density hardness, crystalshape, optical and electrical properties, stability and solubility.Recrystallization solvent, rate of crystallization, storage temperature,and other factors may cause one crystal form to dominate.

As used herein “amorphous” or “non-crystalline” means that the compounddoes not exhibit any substantial peaks in its X-ray diffraction pattern.Typically, non-crystalline materials do not exhibit well defined meltingpoints.

“Solvates” means solvent addition forms that contain eitherstoichiometric or non-stoichiometric amounts of solvent. Some compoundshave a tendency to trap a fixed molar ratio of solvent molecules in thecrystalline solid state, thus forming a solvate. If the solvent is waterthe solvate formed is a hydrate, when the solvent is alcohol, thesolvate formed is an alcoholate. Hydrates are formed by the combinationof one or more molecules of water with one of the substances in whichthe water retains its molecular state as H₂O, such combination beingable to form one or more hydrate.

As used herein, the phrase “pharmaceutically acceptable” refers to thosecompounds, materials, compositions, carriers, and/or dosage forms whichare, within the scope of sound medical judgment, suitable for use incontact with the tissues of human beings and animals without excessivetoxicity, irritation, allergic response, or other problem orcomplication, commensurate with a reasonable benefit/risk ratio.

For the purposes of promoting an understanding of the embodimentsdescribed herein, reference made to preferred embodiments and specificlanguage are used to describe the same. The terminology used herein isfor the purpose of describing particular embodiments only, and is notintended to limit the scope of the present invention. As used throughoutthis disclosure, the singular forms “a,” “an,” and “the” include pluralreference unless the context clearly dictates otherwise. Thus, forexample, a reference to “a composition” includes a plurality of suchcompositions, as well as a single composition, and a reference to “atherapeutic agent” is a reference to one or more therapeutic and/orpharmaceutical agents and equivalents thereof known to those skilled inthe art, and so forth. All percentages and ratios used herein, unlessotherwise indicated, are by weight.

The term “about” is used herein to mean approximately, in the region of,roughly or around. When the term “about” is used in conjunction with anumerical range, it modifies that range by extending the boundariesabove and below the numerical values set forth. In general, the term“about” is used herein to modify a numerical value above and below thestated value by a variance of 20%.

As used in the present disclosure, whether in a transitional phrase orin the body of a claim, the terms “comprise(s)” and “comprising” are tobe interpreted as having an open-ended meaning That is, the terms are tobe interpreted synonymously with the phrases “having at least” or“including at least.” When used in the context of a process the term“comprising” means that the process includes at least the recited steps,but may include additional steps. When used in the context of amolecule, compound, or composition, the term “comprising” means that thecompound or composition includes at least the recited features orcomponents, but may also include additional features or components.

All percentages and ratios used herein, unless otherwise indicated, areby weight. Other features and advantages of the present invention areapparent from the different examples. The provided examples illustratedifferent components and methodology useful in practicing the presentinvention. The examples do not limit the claimed invention. Based on thepresent disclosure the skilled artisan can identify and employ othercomponents and methodology useful for practicing the present invention.

EXAMPLES Example 1 General preparation of phosphonic acid,[[(S)-2-(4-amino-2-oxo-1(2H)-pyrimidinyl)-1-(hydroxymethyl)ethoxy]methyl]mono[3-(hexadecyloxy)propyl]ester (Compound 1) Step 1:Preparation of (S)—N1-[(2-hydroxy-3-triphenylmethoxy)propyl]cytosine(Compound 2)

A slurry of (S)-trityl glycidyl ether (56.4 kg, 178.22 mol), cytosine(18.0 kg, 162.02 mol), and potassium carbonate (2.20 kg, 16.20 mol) indimethylformamide (73.2 kg) was heated to 85-95° C. After 9 hours thereaction mixture was cooled down to 66-70° C. and quenched with toluene(216.0 kg). The resulting slurry was further cooled down to −10 to 5° C.and filtered to collect a solid. This material was washed with toluene(38.9 kg), re-suspended in toluene (168.8 Kg) at 15-25° C., and onceagain filtered.

In order to further remove residual cytosine and process-relatedimpurities a purification cycle was carried out, where the compound waswashed with acetone (36.0 Kg), followed by trituration of the solid inwater/acetone (90.0 kg/54.0 Kg) at 17-22° C., and ending with anotheracetone wash (36.0 Kg). This cycle was repeated several times to improvepurity of the product but should be carried out at least once.

A suspension of the filter cake in acetone (178.9 Kg) at 35-45° C. wasprepared. After 3 hours, the reaction mixture was filtered and theresulting solid was washed with acetone (36.0 Kg) to afford Compound 2(45.0 kg, 65% yield), and dried in vacuo at ≦40° C. for 12 hours. Purityof the compound was determined via HPLC analysis (>99.0%). The compoundwas analyzed by NMR. NMR characterization was consistent with structureof Compound 2.

Step 2: Preparation of Phosphonic acid[[(S)-2-(4-amino-2-oxo-1(2H)-pyrimidinyl)-1-(hydroxymethyl)-2-(triphenylmethoxy)ethyl]methyl]mono[3-(hexadecyloxy)propyl]ester(Compound 3)

A solution of Compound 2 (45.0 kg, 105.26 mol),P-[[[(4-methylphenyl)sulfonyl]oxy]methyl]-,mono[3-(hexadecyloxy)propyl]ester, sodium salt (Compound 4) (66.1 kg,115.79 mol), magnesium tert-butoxide (18.9 kg, 110.53 mol) anddimethylformamide (135.0 kg) was heated at 75-85° C. for 3 hours. Thereaction mixture was cooled down to 25-35° C. and isopropyl acetate (387kg) was added. After completion of the addition the reaction mixture wasfurther cooled down to 15-25° C. and extracted with HCl (aq.; 22.8 kgconc. HCl diluted with 290.8 kg water) and NaCl (aq.; 161.10 kg sodiumchloride dissolved in 606.3 kg water). The resulting organic layer wasvacuum distilled and the concentrate was chased two times with methanolto remove any residual isopropyl acetate to afford Compound 3, which wasused in the next step without further purification.

Step 3: Synthesis of phosphonic acid,[[(S)-2-(4-amino-2-oxo-1(2H)-pyrimidinyl)-1-(hydroxymethyl)ethoxy]methyl]mono[3-(hexadecyloxy)propyl]ester (Compound 1)

Hydrogen chloride gas (11.7 kg, 320.9 mol) was charged to the reactorcontaining a cooled (−5-5° C.) solution of the crude Compound 3concentrate in methanol (276.3 kg). The hydrogen chloride gas wasintroduced below the solvent line in the reaction vessel at a rate wherethe overall reaction temperature remained between −5-15° C. Aftercompletion of the addition, the reaction mixture was maintained at≦15+/−5° C. for 2 hours before being filtered again. The filtrate wasdiluted with water (408.4 kg) and the pH of the reaction mixture wasadjusted to pH=2.3-2.7 with NaOH (aq.; 29.4 kg of a 50% NaOH (aq.)solution was diluted with 337.6 kg water). The resulting solid wascollected via filtration, washed with water (137.6 kg), and re-suspendedin acetone (177.1 kg) at 35-45° C. for 1 hour. The solid was dried at40° C. for 12 hours after a final filtration and acetone wash (2×91.7kg).

The final step involved heating the crude product to 60-70° C. inmethanol (320.8 kg) and then undergoing several slow cooling andfiltration cycles as described below:

The solution was polish filtered and then cooled to 60+/−2° C. Thereaction was maintained at 60+/−2° C. for about 2 hours. The solutionwas then cooled to 50+/−2° C. for about 6 hours, and then to 20+/−3° C.for about 2 hours.

The cooled solution was filtered to collect solids from the solution andthen washed with methanol (91.7 kg).

The solid material was dissolved in methanol (320.8 kg) at 60-70° C.Once dissolved the reaction mixture was stirred at 60+/−1° C. for anadditional 20 minutes before the addition of Compound 1 seed stock(390.0 g). After addition of the seed stock the reaction mixture wasstirred for an additional 2 hours at 60° C. The reaction was slowlycooled to 50+/−3° C. over the next 8 hours while stirring. Once 50° C.was reached, the reaction temperature was maintained for an additional 2hours. A final cooling cycle over 6 hours afforded the reaction mixtureat 20+/−3° C., and stirred for an additional 2 hours. The reactionmixture was filtered to collect solids and the collected solids werewashed with methanol, and dried at ≦40° C. for 24 hours to yieldCompound 1 (41.1 g, 72.4% yield). Purity of the yield was determined byHPLC>99.0%. DSC and XRPD were carried out. The DSC and XRPD data wasconsistent with a composition comprising the morphic Form II.

Example 2 Pilot Crystallizations

About 5 g of Compound 1 was crystallized from methanol. Effects ofdifferent process parameters, including seeding amount (up to 3%), seedtemperature (56° C. to 61° C.), process volume (8.5 to 10), startingmaterial (Form II vs. Form H), excess water content (up to 93:7methanol:water), and agitation rate were evaluated for thecrystallization process. Form II was recovered from each of thecrystallization attempts, including when the process was not seeded withForm II. Each of the crystallizations utilized a slow cooling processand extended slurry periods.

SEM images were collected on several of the crystallization samples.Most samples contained a combination of agglomerates and very thinplate-shaped particles. The agglomeration observed may be due tosecondary nucleation and cementation of fine particles rather thangrowth on existing particles.

Minor differences were observed in the particles and agglomeratesgenerated from the crystallizations with 0.5% seed and the 3% seed(FIGS. 22 and 23). Particle size analysis of these lots showed the 3%seeded sample had smaller d10, d50, and d90 values than the 0.5% asexpected due to the larger number of particles/surface area availablefor crystal growth. The samples that were crystallized using the hydrate(Form H) as the starting material or contained excess water during thecrystallization appeared to generate samples with a lower degree ofagglomeration. The higher water content may have changed the solubilityor induction time and avoided the secondary nucleation and agglomerationobserved in other samples.

The samples prepared with slow agitation still showed significantagglomeration. A seeding step utilizing Form II is performed for controlof form. Optimization of seeding step (size, amount, slurry time, etc.)along with cooling profile avoids the secondary nucleation whichproduces smaller particles, agglomeration, and formation of theundesired Form I. Inclusion of extended slurry times aids in conversionof any Form I that produces to the more stable Form II. Reduction ofwater content to minimal levels avoids conditions that favors theformation of the hydrate.

Example 3 Methods of Synthesis of Compound 1 Form II e.g., CommercialSynthesis

Step 1: Synthesis of(S)-4-Amino-1-(2-hydroxy-3-(trityloxy)propyl)pyrimidin-2(1H)-one(Compound 2)

Compound 2

Representative Material Ratios

4-Aminopyrimidin-2(1H)-one 50.4 kg (453.7 mol) 18.0 kg (162.0 mol)(Cytosine) (S)-Trityl glycidyl ether 158.9 kg (502.2 mol) 56.4 kg (178.2mol) Potassium Carbonate 6.6 kg (47.8 mol) 2.2 kg (16.2 mol)N,N-dimethylformamide 203.3 kg 73.2 kg Toluene 706.4 kg 423.7 kg Acetone958.7 kg 340.9 kg Water 40.1 gal 90.0 kg Yield 126.1 kg to 145.5 kg 45.0to 52.0 kg

Under a nitrogen atmosphere at ambient temperature a reactor was chargedwith cytosine, potassium carbonate, (S)-trityl glycidyl ether, andanhydrous N,N-dimethylformamide. The reaction mixture was heated andmaintained at 85 to 95° C. until reaction was complete and then cooledto 60 to 70° C. The reaction mixture was quenched with toluene thencooled to 0° C. and filtered. The wet solids were washed with toluene,acetone, acetone/water and then acetone. The solids were slurried inacetone at approximately 40° C. then filtered, washed with acetone, anddried under vacuum at approximately 40° C. until the product containedless than 0.5% solvent. Typical yield was approximately 65 to 75% oftheoretical based on Cytosine.

The process for the preparation of Compound 2 contained five in-processchecks to ensure consistent quality of the intermediate: 1. confirm thatthe reaction is complete by measuring the level of cytosine (≦5%; AUC,HPLC); 2. Measuring the remaining cytosine in the isolated and purifiedCompound 2 (level of cytosine≦1%; HPLC, AUC); 3. Measuring the residualsolvent in Compound 2 to ensure that it is ≦0.5%; loss on drying; 4.Confirming that there is ≦0.10% bis-trityl glycidyl ether alkylatedimpurity contained in Compound 2 (AUC, HPLC).

Steps 2A and 2B: Preparation of Phosphonic acid,[[(S)-2-(4-amino-2-oxo-1(2H)-pyrimidinyl)-1-(hydroxymethyl)ethoxy]methyl]mono[3-(hexadecyloxy)propyl]ester (Compound 1)

Representative Material Ratios:

(S)-4-Amino-1-(2-hydroxy-3- 120 kg (238.6 moles) 45.0 kg (105.3 mol)(trityloxy)propyl)pyrimidin-2(1H)- one (Compound 2) Phosphonic acid,P-[[[(4- 151.4 kg(265.3 moles) 66.1 kg (115.8 mol)methylphenyl)sulfonyl]oxy]methyl]-, mono[3- (hexadecyloxy)propyl]ester,sodium salt(Compound 4) Magnesium di-tert-butoxide 43.3 kg (253.9 moles)18.9 kg (110.5 mol) N,N-Dimethylformamide 309 kg 273.0 kg IsopropylAcetate 824.0 kg 387.0 kg Hydorchloric Acid 51.5 kg 22.8 kg Water (forhydrochloric acid 177.2 gal 290.8 kg solution) Sodium Chloride (brine)370.8 kg 161.3 kg Water (for brine solution) 207.7 gal 606.3 kg Methanol1905 kg 852 kg Hydrogen chloride gas 26.8 kg 11.7 kg Water 199 kg 546.0kg Acetone 3,121 kg 537.6 kg Methanol (for recrystallization) 3,254 kg1250 kg Sodium Hydroxide 103.0 kg 29.4 kg Water (for sodium hydroxide337.6 kg solution) CMX001 Seed Stock 390.0 g Yield 87.0 kg to 100.5 kg38.4 kg to 44.3 kg (68.4 to 79.0 mol)

Under a nitrogen atmosphere at ambient temperature an anhydrousN,N-dimethylformamide-rinsed (2×) reactor was charged with anhydrousN,N-dimethylformamide, Compound 2, magnesium di-tert-butoxide, andCompound 4. The reaction mixture was heated and maintained at 75 to 85°C. until complete. The reaction mixture was then cooled to between 25and 35° C., diluted with isopropyl acetate and washed with an aqueoushydrochloric acid solution. The aqueous layer was removed and theorganic layer was washed twice with a sodium chloride solution. Theorganic phase was concentrated and the solvent was switched fromisopropyl acetate to methanol via vacuum distillation. The resultingsolution of intermediate (Compound 3) in methanol was cooled to −5 to 5°C. Hydrogen chloride gas was charged to the reactor and the reaction wasagitated at approximately 15° C. until the reaction was complete. Thereaction mixture was then filtered to remove any insoluble material. Themixture was quenched with water and the pH was adjusted to approximately2.5 with a solution of sodium hydroxide. The resulting solids werefiltered and washed with water. The solids were triturated in acetone atapproximately 40° C., filtered, washed with acetone, and dried. Thesolids were recrystallized from methanol, filtered, and washed withmethanol. The solids were recrystallized a second time from methanol,filtered, and washed with methanol. The solids were dissolved inmethanol and seeded with morphic form II seed stock. The resulting solidwas collected through filtration, washed with methanol, dried undervacuum at approximately 40° C. until there was less than or equal to0.5% residual solvents remaining Typical yield of compound 1 morphicform II: 65 to 75% of theoretical based on Compound 2.

Step 3: Sample Recrystallization Procedure

Representative Material Ratios

3-(hexadecyloxy)propyl 40.0 kg (71.2 moles) 60 g hydrogen((S)-1-(4-amino-2- oxopyrimidin-1(2H)-yl)-3- hydroxypropan-2-yloxy)methylphosphonate (Compound 1) n-heptane 253.0 kg 400 mL Methanol487.9 kg 400 mL + 500 mL Compound 1 seed stock (morphic 360.0 g form II)Part-1. Reprocess from Methanol:

The solid (Compound 1) obtained from step 2B of was dissolved inrefluxing MeOH (450 mL, at around 65° C.) in a 1 L round-bottom flaskand the clear solution obtained was held at around 65° C. for 1 h andcooled to around 61° C.

The contents were stirred at 60° C. for 1 h before gradually cooling to50° C. over an 8 hr period. After holding at 50° C. for 2 hrs, thecontents were further cooled from 50° C. to 20° C. over at least 6 hrs(overnight), stirred at 20° C. for at least 2 hrs and filtered.

The solid obtained was washed with and MeOH (2×25 mL) and dried at 45°C. under vacuum. This process was repeated again two more times.

Part-2A. Reprocess from MeOH-Heptane:

To the hot solution of Compound 1 (60 g) dissolved in refluxing MeOH(360 mL, at around 64° C.) in a 1 L 3-neck round bottom flask was slowlyadded n-heptane (360 mL) by maintaining the internal temp above 50° C.(over 40 min). The contents were held at around 55° C. for 30 min beforegradually cooling to 40° C. over 6 h period.

After stirring at 40° C. for 2 h, the contents were gradually cooledfrom 40° C. to 20° C. over at least 6 h and stirred at 20° C. for atleast 2 h.

The solid obtained was filtered and sequentially washed with n-Heptane(2×20 mL) and MeOH (2×20 mL) and dried under vacuum at ≦45° C. for 12 h,affording white solid. The filtrate was concentrated to dryness and gavean off-white solid (2.6 g).

Part-2B. Form Conversion in MeOH (Process is Performed Three Times, andSeeded with Form II Only for the Last Recrystallization):

The solid obtained from Part-2A was dissolved in refluxing MeOH (450 mL,at around 65° C.) in a 1 L RB flask and the clear solution obtained washeld at around 65° C. for 1 h and cooled to approximately 61° C. andseeded with Compound 1 (1 g).

The contents were stirred at 60° C. for 1 h before gradually cooling to50° C. over an 8 hr period. After holding at 50° C. for 2 hrs, thecontents were further cooled from 50° C. to 20° C. over at least 6 hrs(overnight), stirred at 20° C. for at least 2 hrs and filtered.

The solid obtained was washed with and MeOH (2×25 mL) and dried at 45°C. under vacuum, affording shiny white crystalline solid (57 g).

Approximate yield: 85 to 95% based on Compound 1.

The process for the preparation of Compound 1 contained four in-processchecks to ensure consistent quality of the intermediate: 1. Confirm thatreaction is complete by measuring the level of Compound 2 remaining(should be ≦10.0% (AUC, HPLC)); 2. Determine the content of isopropylacetate (the amount of isopropyl acetate remaining should be ≦5.0% (AUC,GC); 3. Confirm that reaction is complete by measuring the amount ofCompound 3 remaining (should be ≦5.0% (AUC, HPLC)); 4. Determine theamount of residual acetone (the amount of residual acetone is LOD≦0.4%(gas chromatography); 5. Ensure the final product is dry with a residualsolvent check (LOD≦0.4%).

Example 4 Compound 1 Second Purification Procedure

Materials Used

3-(hexadecyloxy)propyl hydrogen ((S)-1-(4- 40.0 kg (71.2 mol)amino-2-oxopyrimidin-1(2H)-yl)-3-hydroxypropan-2-yloxy)methylphosphonate (CMX001) n-heptane 219.0 kgmethanol 521.9 kg CMX001 seed stock (morphic form II) 360.0 kg

Procedure:

Under a nitrogen atmosphere a reactor was charged with CMX001 andmethanol. The mixture was heated to reflux (˜65° C.) and stirred until aclear solution was formed. n-Heptane was added slowly to the reactorover a period of about 40 min while keeping the temperature above 50° C.The temperature was held at about 55° C. for 30 min and then cooled toabout 40° C. over a period of 6 hours. The mixture was stirred at about40° C. for 2 hours, then cooled to 20° C. over a period of 6 hours. Themixture was stirred at 20° C. for 2 hours. The mixture was thenfiltered, washed with n-heptane and methanol, and dried under vacuum at≦45° C.

The resulting solids and methanol were charged to a reactor under anitrogen atmosphere. The mixture was heated to reflux and stirred for atleast one hour. The temperature was adjusted to 60±2° C. and CMX001 seedstock (morphic form II) was added to the reactor. The mixture wasstirred for at least one hour at 60±2° C. and then cooled to 50±2° C.over at least eight hours. The mixture was stirred at 50±2° C. for atleast two hours then cooled to 20±3° C. over at least six hours. Themixture was then stirred at 20±3° C. for two hours, filtered, washedwith methanol, and dried at ≦45° C. until dry (when residual n-heptanelevel is ≦5000 ppm).

The above recrystallization steps are repeated iteratively (e.g., once,twice, three or more times) until the material has reached the desiredpurity. The material is then milled and packaged.

A 5 g sample thus produced was labelled Sample 3 and was subjected toDSC and XRPD analysis. The results are given in FIGS. 20-26.

Yield is approximately 36.0 kg to 39.2 kg (64.1 to 69.8 moles) ofcompound 1 (90 to 98% of theoretical).

The process for the preparation of purified Compound 1 contained twoin-process checks to ensure quality of the intermediate: 1. Confirm theresidual n-heptane is ≦5000 ppm; 2. Confirm that final product is dry(LOD≦0.4%). The residual methanol (≦300 ppm), acetone (≦200 ppm),isopropyl acetate (≦200 ppm), DMF (≦200 ppm), toluene (≦200 ppm),heptanes (≦5000 ppm) and total volatiles other than water (≦6200) wereconducted by GC-HS.

Example 5 Characterization of Compound 1 Form II General

Compound 1 Form II isolated by the methods described in Example 2 wascharacterized by XRPD. The XRPD pattern was consistent with crystallinematerial. The crystalline material was indexed to determine if thecrystal was composed primarily of a single phase. ¹H NMR spectroscopy ofthe crystalline form was consistent with the chemical structure ofCompound 1. The DSC thermogram was carried out, which showed a minorendotherm at ˜43° C. (peak max) followed by overlapping major endothermsat ˜90 and ˜95° C. (peak max). A final endotherm was observed with anonset at ˜196° C. Hot stage microscopy showed no significantobservations prior to flow at ˜189° C. other than some potentialsublimation observed at ˜98° C. Methanol crystallization at a slowercooling profile, but without any stirring also formed Form II. The DSCthermogram of the Form II formed without stirring had a minor endothermat ˜41° C., overlapping endotherms at ˜90 and ˜95° C. (peak max), and afinal endotherm with an onset at ˜200° C.

Crystallization

Crystallization experiments were performed using the Mettler ToledoEasyMax™ 102 with Julabo F26 chiller/circulator. Crystallizations wereperformed in 100-mL glass reactors with turbidity probe, temperatureprobe, and overhead stirring. Experiments performed on the EasyMax™ wereconducted under non-GMP conditions.

Crystallization pilots using EasyMax were run at ˜5 gram scale. In eachof the experiments, the starting material was heated to ˜65° C. toensure complete dissolution. Several process parameters were varied ineach experiment, however a slow cooling (>9 hours) profile was used ineach of the crystallizations. In experiments where seeding was utilized,hand ground seeds of Form II were used. Solids were isolated by vacuumfiltration and dried in a vacuum oven between ˜40° C. and ˜48° C.

Differential Scanning Calorimetry (DSC)

DSC was performed using a TA Instruments Q2000 differential scanningcalorimeter. Temperature calibration was performed using NIST-traceableindium metal. The sample was placed into an aluminum DSC pan, coveredwith a lid, and the weight was accurately recorded. A weighed aluminumpan configured as the sample pan was placed on the reference side of thecell. The method code on the thermogram is an abbreviation for the startand end temperature as well as the heating rate; e.g., −30-250-10 means“from −30° C. to 250° C., at 10° C./min.” See, e.g., FIG. 4.

Hot Stage Microscopy (HSM)

Hot stage microscopy was performed using a Linkam hot stage (FTIR 600)mounted on a Leica DM LP microscope equipped with a SPOT Insight™ colordigital camera. Temperature calibrations were performed using USPmelting point standards. Samples were placed on a cover glass, and asecond cover glass was placed on top of the sample. As the stage washeated, each sample was visually observed using a 10 or 20 objectivewith crossed polarizers and a first order red compensator. Images werecaptured using SPOT software (v. 4.5.9).

Scanning Electron Microscopy (SEM)

SEM was performed using a FEI Quanta 200 scanning electron microscopeequipped with an Everhart Thornley (ET) detector. Images were collectedand analyzed using xTm (v. 2.01) and XT Docu (v. 3.2) software,respectively. The magnification was verified using a NIST-traceablestandard. Each sample was prepared for analysis by placing a smallamount on a carbon adhesive tab supported on an aluminum mount. Eachsample was then sputter coated with Au/Pd using a Cressington 108autoSputter Coater at approximately 20 mA and 0.13 mbar (Ar) for 75 seconds.Each sample was observed under high vacuum using a beam voltage of 5.0kV. The magnification reported on each image was calculated upon theinitial data acquisition. The scale bar reported in the lower portion ofeach image is accurate upon resizing and should be used when making sizedeterminations.

X-Ray Powder Diffraction (XRPD)

XRPD patterns shown in FIGS. 1, 2, 10, 12-14, and 16-17 were generatedusing Pattern Match 2.3.6. XRPD patterns were collected with aPANalytical X'Pert PRO MPD diffractometer either in reflection ortransmission geometry. For reflection geometry, the diffractometer wasconfigured using the symmetric Bragg-Brentano geometry and the incidentbeam of Cu Kα radiation was produced using a long, fine-focus source anda nickel filter. A specimen of the sample was prepared as a thin,circular layer centered on a silicon zero-background substrate.Anti-scatter slits (SS) were used to minimize the background generatedby air. In transmission geometry, the diffractometer used an incidentbeam of Cu radiation produced using an Optix long, fine-focus source. Anelliptically graded multilayer mirror was used to focus Cu Kα X-raysthrough the specimen and onto the detector. A specimen of the sample wassandwiched between 3-μm-thick films and analyzed in transmissiongeometry. A beam-stop, short anti-scatter extension, and anti-scatterknife edge, were used to minimize the background generated by air.Transmission configuration was used most frequently throughout thisstudy. For either configuration, prior to the analysis, a siliconspecimen (NIST SRM 640d) was analyzed to verify the Si 111 peakposition. Soller slits for the incident and diffracted beams were usedto minimize broadening from axial divergence. Diffraction patterns werecollected using a scanning position-sensitive detector (X'Celerator)located 240 mm from the sample and Data Collector software v. 2.2b.

An Anton Paar TTK 450 stage was used to collect in-situ XRPD patterns asa function of temperature. The sample was heated with a resistanceheater located directly under the sample holder, and the temperature wasmonitored with a platinum-100 resistance sensor located in the specimenholder. The heater was powered and controlled by an Anton Paar TCU 100interfaced with Data Collector.

XRPD patterns shown in FIGS. 20, 21, and 25 were collected with aPANalytical X'Pert PRO MPD diffractometer using an incident beam of Curadiation produced using an Optix long, fine-focus source. Anelliptically graded multilayer mirror was used to focus Cu Kα X-raysthrough the specimen and onto the detector. Prior to the analysis, asilicon specimen (NIST SRM 640d) was analyzed to verify the observedposition of the Si 111 peak is consistent with the NIST-certifiedposition. A specimen of the sample was sandwiched between 3-μm-thickfilms and analyzed in transmission geometry. A beam-stop, shortantiscatter extension, and an antiscatter knife edge were used tominimize the background generated by air. Soller slits for the incidentand diffracted beams were used to minimize broadening from axialdivergence. Diffraction patterns were collected using a scanningposition-sensitive detector (X'Celerator) located 240 mm from thespecimen and Data Collector software v. 2.2b. PatternMatch v2.3.6 wasused to create FIG. 25.

Computational Techniques XRPD Indexing

XRPD patterns were indexed using X-Pert High Score Plus (v.2.2.1).Indexing and structure refinement are computational studies which wereperformed under the “Procedures for SSCI Non-cGMP Activities.” Agreementbetween the allowed peak positions and the observed peaks indicated aconsistent unit cell determination. Successful indexing of a patternindicated that the sample was composed primarily of a single crystallinephase. Space groups consistent with the assigned extinction symbol, unitcell parameters, and derived quantities were tabulated in the respectivefigures providing the indexing solution for each form. To confirm thetentative indexing solution, the molecular packing motifs within thecrystallographic unit cells are determined.

XRPD Peak Identification

Under most circumstances, peaks within the range of up to about 30° 2θwere selected. Rounding algorithms were used to round each peak to thenearest 0.1° or 0.01° 2θ, depending upon the instrument used to collectthe data and/or the inherent peak resolution. The location of the peaksalong the x-axis (° 2θ) in both the figures and the tables weredetermined using proprietary software and rounded to one or twosignificant figures after the decimal point. For d-space listings, thewavelength used to calculate d-spacing was 1.541874 Å, a weightedaverage of the Cu-K_(α1) and Cu-K_(α2) wavelengths.

Variability associated with d-spacing estimates was calculated from theUSP recommendation, at each d-spacing, and provided in the respectivedata tables. Per USP guidelines, variable hydrates and solvates maydisplay peak variances greater than 0.2° 2θ and therefore peak variancesof 0.2° 2θ were not applicable to these materials. For samples with onlyone XRPD pattern and no other means to evaluate whether the sampleprovides a good approximation of the powder average, peak tables containdata identified only as “Prominent Peaks.” These peaks were a subset ofthe entire observed peak list. Prominent peaks were selected fromobserved peaks by identifying preferably non-overlapping, low-anglepeaks, with strong intensity.

Particle Size Analysis (PSA)

Particle size data was acquired using a Malvern Instruments Mastersizer2000 equipped with a Hydro2000 μP dispersion unit. Data was collectedand analyzed using Mastersizer 2000 software (v. 5.60) using volumebased measurements. NIST-traceable glass beads were used to qualify theinstrument.

Particle size analysis and Scanning Electron Microscopy (SEM) werecarried out on the isolated crystal Form II. The SEM images showed largeagglomerates along with smaller plate-shaped particles for Compound 1Form II (FIGS. 22-25). Most samples had a bi-modal distribution with amode of small particles at ˜6-10 μm and a larger mode at ˜60-160 μm.Sizes of the particles differed depending on the type of sample. Threesamples were characterized by SEM and particle size analysis. Theseincluded: the starting material; the methanol recrystallized material;and the comilled material from a ˜45 kg recrystallization batch.

The starting material contained agglomerates (˜100 μm) composed ofsmaller plates. The particle size distribution for the starting materialwas bi-modal. The methanol recrystallized sample contained largerprimary particles, some agglomeration without cementation and a singleparticle size mode. The comilled sample was similar to the methanolrecrystallized sample but showed a slightly smaller particledistribution suggesting that only minor particle attrition occurredduring the milling step. The SEM images also suggested little attritionbased on the morphology of the particles.

Example 6 Characterization of Drug Product Samples

XRPD and DSC analysis were completed on two samples of Compound 1tablets. Prior to analysis, the tablets were lightly ground using amortar and pestle. The two tablet samples showed very similar XRPDpatterns (see FIGS. 16 and 17) indicative of crystalline material withdiffuse scatter potentially from one or more excipients. Comparison withForm I, Form II, and Form H showed several peaks consistent with Form IIin both tablet samples (FIG. 14). The DSC thermograms (FIGS. 18 and 19)for both tablet samples showed overlapping minor endotherms at ˜90° C.and ˜95° C. (peak max) and a major endotherm with an onset at ˜165° C.The two minor endotherms were consistent with endotherms observed forForm II; however Form II also showed a large endotherm at about 196° C.that was not observed in either sample. Compare FIGS. 18 & 19 with FIG.4.

Example 7 Stable Form Screening of Compound 1 Form II

In order to aid in the design of solid form screening experiments,solubility estimates were completed in various solvent systems usingForm II at ambient and elevated temperature. Generally poor solubilitywas observed in each of the solvents tested. Solubility greater than 5mg/mL was observed in Trifluoroethanol. Slurries were set up in avariety of solvent systems to determine the stable form and thepotential of Compound 1 to form stable solvates. The slurries wereprepared using Form II and each was seeded with Form I. Each of theslurries was stirred for about two weeks and most of the slurries wererun at room temperature although a few were run at sub-ambient andelevated temperatures (˜45° C.). Form II was recovered in each of theanhydrous solvent systems. A few samples did show a minor amount of FormI in Form II likely due to poor conversion kinetics due to limitedsolubility. Each of the slurries in aqueous solvent systems was found tohave converted to the hydrated form, Form H. No evidence of new forms,including a potential methanol solvate, was observed. Interconversionstudies starting with a mixture of Form I and Form II were also run inmethanol at room temperature and ˜45° C. Solids recovered from theseexperiments were found to be consistent with Form II suggesting Form IIis the most stable anhydrous form between room temperature and ˜45° C.

Example 8 Methanol Solubility and Metastable Zone

Solubility Determination: Aliquots of test solvents or solvent mixtureswere added to weighed samples of Compound 1. Samples were sonicated asneeded between additions to facilitate dissolution. Complete dissolutionof the test material in each solvent was determined by visualinspection. The solubility was estimated based on the total volume ofsolvent needed to provide complete dissolution. The actual solubilitymay be greater than the value calculated due to the incremental additionof solvent and kinetics of dissolution of the material. The solubilityis expressed as “less than” if dissolution did not occur during theexperiment, or “more than” if dissolution occurred after the addition ofthe first aliquot. See Table 2 and discussion under “Solubility” sectionin the “Detailed Description” of the present disclosure.

EQUIVALENTS

The invention can be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The foregoingembodiments are therefore to be considered in all respects illustrativerather than limiting on the invention described herein. Scope of theinvention is thus indicated by the appended claims rather than by theforegoing description, and all changes that come within the meaning andrange of equivalency of the claims are intended to be embraced therein.

What is claimed is:
 1. A method of preparing the morphic Form II ofcompound 1:

comprising: (a) recrystallizing compound 1 three times from methanol;(b) recrystallizing compound 1 once from n-heptane and methanol; and (c)recrystallizing compound 1 again from methanol.
 2. The method of claim1, wherein step (c) is carried out with seeding with an amount ofmorphic Form II of Compound
 1. 3. The method of claim 2, wherein themorphic Form II is seeded at an amount of about 0.5% wt/wt, about 3%wt/wt, or about 7% wt/wt.
 4. The method of claim 1, wherein the firstthree recrystallizations from methanol each comprise: (a) dissolvingCompound 1 in methanol at about 60-70° C. and stirring for about fiveminutes; (b) cooling to 60° C. and stirring for about one hour; (c)cooling to 50° C. over about six hours; (d) cooling to 20° C. over abouttwo hours and stirring for two hours; and (e) filtering the resultingsolid.
 5. The method of claim 1, wherein step (b) comprises: (a)dissolving compound 1 in refluxing MeOH; (b) adding n-heptane whilemaintaining the internal temp above about 50° C. over about 40 minutes;(c) maintaining the solution at about 55° C. for about 30 minutes; (d)cooling the solution to about 40° C. over about six hours; (e) stirringthe solution at about 40° C. for about two hours; (f) cooling thesolution to about 20° C. over about at least six hours and stirring atabout 20° C. for about at least two hours; and (g) filtering theresulting solid.
 6. The method of claim 5, further comprising washingthe solid with n-hexane and methanol.
 7. The method of claim 2, whereinstep (c) comprises: (a) stirring a solution of compound 1 at about 60°C. for about one hour; (b) cooling the solution to about 50° C. overabout eight hours; (c) holding at about 50° C. for about two hours; (d)cooling the solution to about 20° C. over about six hours; (e) stirringthe solution for about two hours at about 20° C.; and (f) filtering theresulting solid.
 8. The method of claim 7, further comprising seedingthe solution with a morphic Form II of Compound 1 after step (a).
 9. Themethod of claim 1, wherein the resulting morphic Form II of Compound 1is anhydrous.
 10. A pharmaceutical composition comprising the morphicForm II of Compound 1 prepared by the method of claim
 1. 11. A method ofsynthesizing Compound 2,

comprising the steps: (a) heating a mixture of (S)-trityl glycidylether, cytosine, potassium carbonate, and N,N-dimethylformamide (DMF);(b) cooling the reaction mixture and quenching with toluene to produce aslurry; (c) cooling the slurry of step (b), filtering to give a solidand washing the solid with toluene; (d) slurrying the solid of step (c)in toluene, filtering to give a solid, and washing the solid withacetone; (e) triturating the solid of step (d) in water/acetone; and (f)filtering to give a solid filter cake.
 12. The method of claim 11,further comprising washing the solid filter cake with acetone andsuspending the solid filter cake in acetone.
 13. The method of claim 12,further comprising removing the acetone from the suspended filter cakeand drying the filter cake in vacuo.
 14. The method of claim 11,comprising repeating steps d-e at least once.
 15. The method of claim12, wherein steps d-e are repeated twice.
 16. The method of claim 11,wherein the purity of compound 2 is at least 91% (wt/wt).
 17. A methodof preparing morphic Form II of Compound 1, comprising the step ofcombining (S)—N¹-[(2-hydroxy-3-triphenylmethoxy)propyl]cytosine Compound2, and P-[[[4-methylphenyl)sulfonyl]oxy]methyl]-,mono[3-(hexadecyloxy)propyl]ester, sodium salt Compound 4 with magnesiumtert-butoxide, and a suitable organic solvent and crystallizing frommethanol to produce morphic Form II of Compound 1, wherein the morphicForm II is not a hydrate.
 18. Morphic Form II of Compound 1 in stablecrystalline form.
 19. A composition comprising morphic Form II ofCompound 1

and Compounds A, B, C, and D:


20. The composition of claim 19 comprising greater than 90% wt/wtcompound
 1. 21. The composition of claim 19 comprising less than 10%wt/wt total Compounds A, B, C, and D.